The HECT family ubiquitin ligase NEDD4L and DUB USP20 also participate in autophagy termination. During prolonged starvation, NEDD4L catalyzes the K27 and K29 ubiquitination on ULK1 [ 58 ], whereas the interaction between USP20 and ULK1 is attenuated [ 43 ].

Both mechanisms lead to downregulation of ULK1 protein level. Thus, multiple E3 ligases and DUB act in concert to limit ULK1 protein abundance, thereby contributing to autophagy termination.

Importantly, the ULK1 mRNA is consistently present and its translation is induced when mTOR is reactivated by the release of building blocks from the autolysosome. This mechanism allows the recovery of ULK1 protein level for the next run of autophagy induction [ 58 ].

Autophagy was originally considered as a nonselective bulk degradation process, but numerous studies have later reported the selective degradation of various cellular organelles or substances via autophagy mechanism, including mitochondria, ER, peroxisome, lipid droplet, ribosome, midbody, nucleus, protein aggregate, and specific pathogens [ 59 ].

In theory, selective autophagy should result in a more specific removal of damaged or harmful cellular components and thus could be more important in disease prevention than bulk autophagy. To achieve selectivity, the cargos are often linked to LC3 family proteins directly or indirectly via ubiquitin-dependent or independent mechanisms.

This review focuses only on the ubiquitin-dependent selective autophagy. Different from the bulk autophagy where protein ubiquitination often plays a modulating role, protein ubiquitination in many types of selective autophagy serves as a mark for cargo recognition and a signal for process initiation.

Ubiquitinated proteins generated on the surface of cargos are responsible for the recruitment of specific autophagy adaptor proteins also known as autophagy receptors , such as p62, OPTN, NBR1, NDP52, and TAX1BP1 [ 60 , 61 ].

Since these autophagy adaptors possess both ubiquitin-binding domain and LC3-interacting region LIR , they function as bridges to recruit LC3 to the cargos.

Certain autophagy adaptor, such as NDP52, also recruits upstream autophagy initiating complex to the cargos [ 62 , 63 ]. In this way, autophagy machinery generates autophagosome to specifically engulf the cargos. Below, we discuss the role of ubiquitination in the initiation and regulation of several types of selective autophagy Fig.

Ubiquitin-dependent selective autophagy. Summary of the molecular mechanisms of major types of selective autophagy using protein ubiquitination as a mark of the cargo. The E3 ligases and DUB involved in generating or removing the ubiquitin chain and the autophagy adaptors used to link ubiquitinated cargos to LC3 are indicated.

The best studied ubiquitin-dependent selective autophagy mechanism is mitophagy, in which the protein kinase PINK1 and E3 ligase Parkin play a key role in building the ubiquitin chains on the outer surface of damaged mitochondria. Upon mitochondria damage, PINK1 is stabilized on mitochondria membrane to recruit Parkin [ 64 , 65 , 66 ] and phosphorylates the S65 residue on both ubiquitin and the UBL domain of Parkin, which act in concert to activate Parkin on mitochondria [ 67 , 68 , 69 ].

Parkin in turn catalyzes the ubiquitination of numerous mitochondrial outer membrane proteins [ 70 , 71 ]. Recent studies indicate that these ubiquitinated proteins not only facilitate the recruitment of autophagy adaptors but also serve as PINK1 substrates to establish a feedforward mechanism for reinforcing the PINK1-Parkin pathway [ 68 , 72 ].

Quantitative proteomic study identified numerous mitochondrial proteins whose ubiquitination is dependent on Parkin [ 73 ]. Furthermore, multiple ubiquitin chain types, such as K6, K11, K48 and K63 are generated following mitochondrial depolarization [ 68 ].

It is generally believed that the identity of the substrates is less important than the density of ubiquitin chains on mitochondria to determine the onset of mitophagy [ 74 ]. Consequently, autophagy adaptors are recruited to the damaged mitochondria.

CRISPR-mediated knockout analysis on HeLa cells revealed that OPTN, NDP52 and TAX1BP1 are redundantly required for mitophagy, with OPTN playing the most prominent role [ 75 ]. OPTN further recruits TBK1 to promote mitophagy through a feedback mechanism [ 76 , 77 ].

Nevertheless, other study indicated the crucial role of p62 in Parkin-dependent autophagy in mouse macrophages and embryonic fibroblasts [ 78 , 79 ]. It is unclear whether this discrepancy is owing to the difference in the relative abundance of these adaptors in different cell types.

Besides Parkin, mitophagy can be regulated by other factors that influence on the ubiquitination of mitochondrial membrane proteins.

USP30, a transmembrane DUB localized on the mitochondrial outer membrane, antagonizes the function of Parkin by removal of ubiquitin chains from mitochondria [ 80 ]. Interestingly, USP30 undergoes a Parkin-dependent monoubiquitination and proteasomal degradation, thus establishing a feedforward mechanism for Parkin to promote mitophagy.

Additionally, E3 ligases other than Parkin that target mitochondrial fusion and fission machineries [ 81 , 82 ] can also regulate mitophagy, as damaged mitochondria need to go through a fission process to be enclosed into the autophagosome [ 83 ].

Peroxisomes are ubiquitous organelles involving in modulation of metabolic responses and redox regulation [ 84 ]. In mammals, damaged peroxisomes are removed through ubiquitin-dependent selective autophagy pathway [ 85 ]. Consistently, an increase in ubiquitinated proteins on the surface of peroxisomes induces pexophagy.

Peroxisome membrane proteins PEX5 and PMP70 are targeted for monoubiquitination under stressed conditions through the peroxisome E3 ligase PEX2 [ 86 ]. As to the autophagy adaptors, p62 and NBR1 act in a cooperated fashion to link ubiquitinated peroxisome to autophagic machinery [ 85 , 87 ].

Although bulk autophagy and selective autophagy require the fusion with lysosome for autophagic flux, damaged lysosome is itself removed by an autophagic process called lysophagy. Lysophagy utilizes a ubiquitin-dependent selective autophagy mechanism, as ubiquitinated proteins, p62, and LC3 are all found on the surface of damaged lysosomes [ 88 , 89 ].

The damaged lysosome membranes are also decorated with galectin-3 [ 89 ], which is presumably due to the exposure of the luminal proteins to the cytosol side following membrane rupture. Recent study indicates that FBXO27, a membrane localized substrate adaptor of Cul1 ubiquitin ligase, catalyzes the ubiquitination of N-glycoproteins exposed to the damaged lysosome, thereby facilitating the recruitment of autophagy adaptor p62 [ 90 ].

In addition to cellular organelles, ubiquitin-dependent selective autophagy is also exploited to eliminate intracellular pathogens such as Salmonella, Listeria, and Mycobacterium , a process called xenophagy [ 91 ].

In the host cells, these pathogens are quickly marked by ubiquitin chains on their surface. Multiple host E3 ligases are reported to ubiquitinate pathogens. For instance, Smurf1 and Parkin are involved in the ubiquitination of M. tuberculosis [ 92 , 93 ]. LRSAM1, ARIH, and HOIPI complex are responsible for Salmonella ubiquitination [ 23 , 94 , 95 ].

Of note, the ubiquitin chain types generated by these E3 ligases are different. While LRSAM1 generates K6 and K27 chains, ARIH and HOIP1 form K48 chain and M1 chain, respectively.

These different ubiquitin chains are clustered to form distinct foci on bacteria surface [ 96 ]. The M1 chain specifically recruits OPTN, whereas the recruitment of p62 and NDP52 to bacteria is independent of M1 chain, demonstrating their non-redundant functions [ 97 ].

In addition to inducing xenophagy, the M1 chain on bacteria activates NF-kB pathway to promote proinflammatory cytokine secretion, thereby inhibiting bacteria proliferation [ 96 , 97 ].

Aggrephagy is induced in response to various proteotoxic conditions, such as inhibition of proteasome or chaperons and interference with productive translation, in which aggregates of ubiquitinated proteins are observed [ 98 ]. Formation of such aggregates requires p62 [ 99 ].

Recent studies indicate that p62 drives the aggregate formation via a process called liquid-liquid phase separation [ 61 , ].

In addition to the ubiquitin binding domain UBA , p62 contains a oligomerization domain PB1. Oligomerization of p62 allows a high-avidity binding of ubiquitinated proteins via UBA domain and finally condenses the ubiquitinated proteins into larger structures. Subsequently, P62 tethers LC3 to the condensates through its LIR to facilitate a selective sequestration of ubiquitin condensates to the autophagosome.

Other autophagy adaptor, such as NBR1, can also contribute to the condensation by interacting with p62 [ ]. Since ubiquitinated proteins can also be targeted to undergo proteasomal degradation, one intriguing question is how to distinguish the autophagy fate from proteasome fate.

Although pmediated condensation may be a determining factor to direct ubiquitinated proteins to the autophagy pathway, it is worth noting that p62 can also function as a direct adaptor to recruit ubiquitinated proteins to the proteasome in cytosol or nucleus [ , ].

Another possibility for determining the fate of ubiquitinated protein is the quality of ubiquitin chains. It is thought that Kubiquitinated proteins are degraded by proteasome, whereas K63 chain modified proteins are substrates of aggrephagy. However, M1, K63, and K48 chains can all trigger phase separation in vitro via binding to p62, albeit with a lower efficiency than the K48 chain [ 61 , ].

Perhaps the nature of aggrephagy substrates do not have much difference from those of the proteasome substrates and, rather, the high concentration of ubiquitin chains determines the aggrephagy fate by favoring a pmediated phase separation [ ].

The most well-known neurodegenerative disease associated with defects in ubiquitin-mediated autophagy is PD, which is the second most common late-onset neurodegenerative disease resulted from the loss of dopaminergic neurons in the substantia nigra pars compacta.

Mutations in genes encoding either PINK1 or Parkin are associated with autosomal recessive forms of PD [ ]. Mice deficient in either Parkin or PINK1 exhibit mitochondrial impairments, but most of them cannot recapitulate the prime features of human PD, that is, loss of dopaminergic neurons [ , ].

A recent study generated by Parkin homozygous knockout in the background of mice with the expression of a proof-reading defective mtDNA polymerase called mutator mice. The combination of Parkin knockout and mtDNA mutation leads to the loss of dopaminergic neurons selectively in the substantia nigra and motor defect [ ].

This genetic evidence, in conjunction with the mitochondrial dysfunction found in brain and other organs of PD patients [ ], point out the importance of mitophagy in PD etiology. A recent study uncovered a link of ubiquitin-mediated autophagy regulation to various polyQ diseases.

Ataxin 3 is a polyQ-containing DUB and its polyQ expansion is associated with SCA type 3, in which neurodegeneration occurs in the striatum and cerebellum [ ]. Interestingly, the normal function of ataxin 3 is to remove the polyubiquitin chain from Beclin-1, leading to its stabilization [ 38 ].

With this function, ataxin 3 is required for starvation-induced autophagy. Importantly, several proteins with expanded polyQ repeats, including ataxin 3 itself, can compete with ataxin 3 for binding Beclin-1, in a polyQ length-dependent fashion.

Furthermore, although ataxin 3 with expanded polyQ repeats elicits higher binding affinity to Beclin-1, it is defective in removing ubiquitin chain from Beclin Thus, these findings identify a link of ataxin 3 to autophagy regulation and, more importantly, suggest that impairment of Beclinmediated autophagy accounts for one mechanism of polyQ repeat-associated neurodegenerative diseases.

As described above, ubiquitin serves as a tag to facilitate the autophagic degradation of intracellular pathogens xenophagy and a number of ubiquitin E3 ligases are involved in the addition of such tag. Since autophagy core machinery is also required for the xenophagy process, regulators that affect ubiquitin-dependent turnover of autophagic core factors could also control xenophagy.

For instance, RNF, which targets Beclin-1 for ubiquitination and degradation, promotes Listeria monocytogenes proliferation and distribution in cell and mouse models [ 32 ]. Nevertheless, it should be noted that the bulk autophagy could elicit housekeeping function to restrict inflammation, thereby favoring pathogen survival [ 91 ].

The balance between selective autophagy and anti-inflammation could determine the outcome of infection and immunological functions. One example for ubiquitination-mediated balance of anti-infection arm and anti-inflammation arm lies in USPdepedent Beclin-1 deubiquitination [ 39 ].

On one hand, this deubiquitination stabilizes Beclin-1 to favor autophagy-dependent pathogen clearance. On the other hand, the stabilized Beclin-1 binds to the CARD domain of MAVS to prevent MAVS-RIG-I association, thereby inhibiting type I interferon production and anti-viral immunity.

Autophagy is important in controlling hepatocyte lipid metabolism to maintain normal liver functions [ ]. Autophagy deficiency by ATG7 knockout aggravates liver steatosis induced by high fat diet and promotes the development of liver adenoma [ ].

Conversely, liver steatosis impairs autophagy through ATG7 downregulation [ ]. One important function of autophagy to regulate lipid metabolism is the turnover of lipid droplets via a selective autophagy process called lipophagy [ ]. Similar to other selective autophagy processes, lipophagy requires certain core autophagic factors.

A recent study reveals an inhibitory role of HUWE1-mediated WIPI2 degradation in lipid droplet turnover in the liver, leading to the accumulation of liver neural lipids [ 48 ].

Besides liver disease, ubiquitin-mediated autophagy regulation is implicated in other metabolic syndromes. For instance, failure of autophagy termination by KLHL20 deficiency potentiates muscle atrophy in diabetes mouse model [ 57 ].

Autophagy plays complex roles in cancer, which may depend on the different stages of cancer development. In the tumor initiating stage, autophagy suppresses carcinogenesis. However, once tumor is formed, tumor cells exploit the autophagic process for them to survive in the harsh environments [ 17 ].

The impact of ubiquitin-mediated autophagy regulation on tumor formation and progression is poorly studied. A recent study reported that the Smurf1-induced UVRAG ubiquitination promotes not only autophagosome maturation but hepatocellular carcinoma HCC growth [ 56 ].

Furthermore, phosphorylation of UVRAG at S, which disrupts Smurf1 binding, correlates with poor survival of HCC patients. These findings support a tumor suppressive role of autophagy in HCC.

In this review, we discussed the impact of protein ubiquitination in autophagy regulation. The initiation and nucleation steps of autophagosome formation are most prevalently regulated by ubiquitination, meaning that ubiquitination controls the onset of autophagic process in response to various stressed conditions.

Nevertheless, later steps of autophagosome formation and autophagosome maturation are also subjected to ubiquitin-mediated regulation. Furthermore, ubiquitin-mediated protein turnover has been used as a prime mechanism for autophagy termination under prolonged stress conditions, thereby preventing the detrimental effect of excessive autophagic degradation.

The pleiotropic role of protein ubiquitination in autophagy regulation highlights the tight crosstalk between the two major cellular degradation machineries.

Dysregulation of ubiquitin-mediated autophagy process has been implicated in many disease states, such as neurodegeneration, infectious diseases, liver diseases and metabolic syndromes. With the important role of autophagy in maintaining normal physiology and homeostasis, it is expected to uncover further linkages between dysregulation of ubiquitin-mediated autophagy pathways and various human diseases, especially for age-related diseases.

In this regard, targeting of these pathways by modulating the activity of E3 ligase or DUB could be exploited as a strategy for disease intervention and has been an area receiving considerable attention. For example, the small molecular inhibitor of USP10 and USP13, called spautin-1, is capable of antagonizing the ubiquitination and degradation of Beclin-1 and p53, two tumor suppressor proteins, and therefore is a promising anti-cancer agent [ 37 ].

In the future, an improved understanding of how ubiquitin-mediated autophagy regulation contributes to the pathology of human diseases and the development of less toxic and more specific agents will benefit more patients.

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Although NIX-dependent mitophagy was predominantly studied in reticulocytes, NIX-dependent mitophagy might be important for other cell types as well [for example, see Esteban-Martínez et al.

Autophagy of peroxisomes, pexophagy, is a selective degradation process of peroxisomes during which the UPS and autophagy mechanisms work in collaboration.

Peroxisomes are responsible of a number of cellular functions, including fatty acid oxidation, purine metabolism and phospholipid synthesis Wanders et al. Several peroxisomal enzymes are involved in redox regulation due to their dual functions in the generation and scavenging of reactive oxygen and nitrogen species.

Therefore, peroxisome biogenesis and degradation must be tightly regulated in order to control peroxisome size, number and function Du et al. Moreover under stress conditions such as hypoxia, oxidative stress, starvation or conditions causing UPS defects, pexophagy is upregulated.

During pexophagy, a number of peroxisomal membrane proteins, including peroxins and PMP70 become ubiquitylated Kim et al. PEX2-PEXPEX12 complex serves as an E3 ligase at least for two well studied peroxisome proteins, PEX5 and PMP For example, PEX2 overexpression or amino acid starvation activated the ubiquitylation of PEX5, and another peroxisomal membrane protein, PMP70, and led to peroxisome degradation Sargent et al.

Moreover in response to oxidative stress, ATM was recruited onto peroxisomes through physical interaction with PEX5 and promote its ubiquitylation. Inactivation of mTORC1 in a TSC2-dependent manner and stimulation of ULK1 phosphorylation by ATM, potentiated pexophagy Zhang J.

On the other hand, AAA ATPase complex PEX1, PEX6, and PEX26 was shown to extract ubiquitylated PEX5 from peroxisomal membranes and regulate pexophagy Carvalho et al.

Both NBR1 and p62 were shown to be recruited onto peroxisomes during pexophagy. Yet, NBR1 was a major pexophagy receptor in a number of contexts, and p62 increased the efficiency of NBR1-dependent pexophagy through direct interaction with the latter Deosaran et al.

Altogether, these findings underline the importance of ubiquitylation for the selective degradation of peroxisomes by autophagy. FIGURE 8. Selective removal of peroxisomes by autophagy utilizes ubiquitylation as signal.

In addition to major cellular organelles, autophagy was implicated in the clearance of ribosomes. Although ribosomes can be degraded in a non-specific manner during non-selective autophagy, a special form of selective autophagy is activated under various stress conditions, and the process is called ribosomal autophagy or ribophagy.

On the other hand, mRNA protein complexes that are stalled during translation form stress granules, and their clearance requires both the UPS and autophagy. In the mammalian system, in addition to mTOR inhibition, oxidative stress, induction of chromosomal mis-segregation, translation inhibition and stress granule formation were all shown to induce ribophagy An and Harper, Ubiquitylation of ribosomes was observed under ER stress-inducing conditions Higgins et al.

Yet, individual ribosomal proteins were indeed shown to be a target of the UPS Wyant et al. NUFIP1-ZNHIT3 proteins were identified as novel ribophagy receptors that directly connected ribosomes to LC3 and autophagy, yet whether ubiquitylation is a prerequisite for ribophagy needs to be clarified by future studies Wyant et al.

FIGURE 9. Ubiquitylation primes ribosomes and stress granules for proteasomal degradation and autophagic elimination.

Stress granules are composed of actively accumulated non-translating mRNA ribonucleoprotein complexes Protter and Parker, Proteins that accumulated in the stress granules, include stalled 40S ribosomal units and various translation initiation factors [e.

G3BP1 and TIA-1 are also among the proteins that contribute to stress granule formation Kedersha et al. Moreover, an interplay between G3BP1 and Caprin1 proteins and the DUB protein USP10 was shown to regulate stress granule formation Kedersha et al.

HDAC6 protein was a component of stress granules as well Seguin et al. Endoplasmic reticulum ER stress is one of the conditions under which both the UPS and autophagy pathways are being activated. Abnormalities in calcium homeostasis, oxidative stress and conditions leading to protein glycosylation or folding defects etc.

ER stress might be very destructive for cells, therefore ER-specific stress response pathways such as the unfolded protein response UPR and the ER-associated degradation ERAD pathways were evolved.

Both pathways are directly or indirectly connected to the UPS and autophagy. In mammalian cells, accumulation of unfolded proteins in the lumen of the ER result in the activation of stress responses.

PERK activation leads to the phosphorylation of the α subunit of the translation initiation factor, eIF2α, which inhibits the assembly of the 80S ribosome and cap-dependent protein synthesis, while allowing cap-independent translation of the stress response genes such as ATF4.

Activation of IRE1 and ATF6 promotes transcription of other stress response genes. IRE1-mediated processing generates a splice-form of the XBP1 mRNA, resulting in the production of a transcription factor that upregulates chaperones and other relevant genes. Due to a decrease in the protein load in the ER and an increased folding capacity, the UPR facilitates recovery from stress.

In case of failure, the UPR sensitizes cells to programmed death mechanisms. FIGURE Crosstalk between the UPS and autophagy systems during ER stress and ERAD. Components of the UPR were subject to active regulation by the UPS.

For example, SCF component E3 ligase βTrCP was shown to lead to the ubiquitylation ATF4 following its phosphorylation Lassot et al.

CHOP stability was regulated by the UPS and p and cIAP were responsible for CHOP ubiquitylation and degradation counterbalancing its upregulation during ER stress Qi and Xia, ; Jeong et al. Another UPR component, IRE1 was identified as a ubiquitylation target of the E3 ligase CHIP during ER stress.

Ubiquitylation IRE1 inhibited its phosphorylation, perturbed its interaction with TRAF2, and attenuating JNK signaling Zhu et al. Under stress conditions, translation of XIAP, an E3 ligase protein and an inhibitor of apoptosis was downregulated in a PERK-eIF2α-dependent manner.

In the same context, ATF4 may promote ubiquitylation and degradation of XIAP, leading to sensitization of cells to ER stress-related cell death Hiramatsu et al. Conversely, activation of PERK-eIF2α axis might also show opposing effects through induction of other IAP proteins, cIAP1 and cIAP2, and counter balance cell death induction signals Hamanaka et al.

Endoplasmic reticulum stress was shown to trigger autophagy, and ER-related stress response mechanisms were involved in the process. PERK-mediated phosphorylation of eIF2α and resulting ATF4 and CHOP activation, were associated with the transcription of genes such as ATG5, ATG12, Beclin1, ATG16L1, LC3, p62 and TSC2 activator, hence mTOR inhibitor REDD1 Whitney et al.

Moreover, CHOP downregulated BCL2 binding Mccullough et al. TRB3, an AKT inhibitor protein, was also described as a target of CHOP Ohoka et al. In addition, IRE1 activation resulted in the recruitment of ASK1 by the adaptor TRAF2 and the outcome was the activation of JNK and p38 kinases Nishitoh et al.

BCL2 is one of the targets of JNK, its phosphorylation by the kinase resulted in destabilization the inhibitory BCL2-Beclin1 complex, stimulating autophagy Bassik et al. On the other hand, in its unspliced form, IRE1 splicing target XBP1, in its unspliced form was shown to target the autophagy activator FOXO1 for degradation by the UPS Vidal et al.

Endoplasmic reticulum is a major calcium store in cells, and calcium release to cytosol was observed during ER stress. In addition to problems with SERCA refill pumps and leakiness of membranes during stress, upregulation of ERO1-α by CHOP resulted in an IP3-mediated calcium release Li et al.

Calcium binding protein calmodulin senses the cytosolic increase in the concentration of the ion, and bind to calmodulin-regulated kinases such as CaMKII and DAPK1, modulating their activity.

Activated CaMKII was shown to stimulate autophagy through AMPK phosphorylation and activation Høyer-Hansen et al. In addition, calmodulin-binding and PP2A-mediated dephosphorylation was necessary for the activation of the autophagy-related kinase DAPK1 Gozuacik et al.

DAPK1 could directly phosphorylate Beclin1 on the BH3-domain, resulting in the dissociation of Beclin1 from the BCL2-Beclin1 complex and allowing it to stimulate autophagy Zalckvar et al.

Proteins that accumulate in the ER are degraded by the ER-associated degradation ERAD system. ERAD mediates transport, extraction and ubiquitylation of proteins that cannot be salvaged and target them for degradation in proteasomes.

In mammalian cells, ER membrane-resident complexes containing E3 ligases such as HRD1 and GP78, and other regulatory components such as EDEM1, SEL1L, ERManI, and HERP control the ERAD pathway.

DUB proteins, including YOD1, USP13, USP19, and Ataxin-3 were implicated in the control of client protein ubiquitylation and ERAD substrate modulation Zhong and Pittman, ; Bernardi et al.

ER-associated degradation regulators and therefore ERAD might be controlled by the UPS and autophagy pathways. For example, E3 ligase Smurf1 was found to be downregulated during ER stress, resulting in the accumulation of its direct ubiquitylation target WFS, which is a stabilizer ER-related E3 ligase HRD1 Guo et al.

Smurf1 was also involved in selective bacterial autophagy Franco et al. On the other hand, while the ERAD complex component HERP protein was degraded by the UPS Hori et al.

An ER-localized E3 ligase synoviolin protein was shown to ubiquitylate HERP protein and control its degradation by proteasome Maeda et al. Yet, other ERAD-related components, EDEM1 and Derlin2 as well as ubiquitylated EDEM1 proteins colocalized with cytoplasmic aggregates and autophagy receptors p62 and NBR1, they were degraded by selective autophagy Le Fourn et al.

ERManI, a mannosidase that is responsible for priming ER-resident glycosylated proteins for degradation, was described as an accelerator of the ERAD pathway and clearance of clients by the UPS.

But, following proteasome inhibition and subsequent ER stress, ERManI colocalized with LC3 and degraded in an autophagy-dependent manner Benyair et al. All these findings point out to the presence of important junctions and coregulation nodes between the UPS and autophagy in the context of ER stress.

Additionally, ERphagy, the autophagy of portions of the ER, was implicated in the recovery from ER stress and control of ER size, but this mechanism was so far described as a ubiquitin-independent process Schuck et al.

Several transcription factors that are regulated by the UPS, including p53, NFκB, HIF1α, and FOXO, have been implicated in the control of autophagy.

In general, these factors were shown to directly activate transcription of key autophagy genes under stress conditions. Some autophagy proteins such as LC3 are consumed in the lysosome following delivery, and during prolonged stress, cellular levels of these proteins are sustained by mechanisms, including transcription.

On the other hand, regulation of the transcriptional activity NRF2 involves a special crosstalk between the two systems. In this section, we will summarize molecular details of transcription regulation by the UPS and autophagy.

P53, a guardian of the genome, is one of the well-known transcriptonal regulators that has a dual role in autophagy depending on its intracellular localization. Accumulating p53 protein activates transcription of several stress- and death-related genes, including autophagy-related genes PRKAB1 , PRKAB2, TSC2 , ATG2, ATG4, ATG7 , ATG10 , ULK1 , BNIP3, DRAM1, and SESN2 Crighton et al.

On the other hand, a cytosolic form of p53 was shown to inhibit AMPK and activate the mTOR pathway. Additionally, another E3 ligase, NEDD was shown to control MDM2 stability and p53 activation Xu et al.

In addition to MDM2, another E3 ligase, PIRH2, was able to ubiquitylate p53 to control its cellular stability Shloush et al. NF-κB is a well studied transcriptional regulator of autophagy. As a result of its association with IκB, NF-κB is found in an inactive state in the cytosol.

In response to agonists, IκB was reported to be ubiquitylated and subsequently degraded by the UPS. Phosphorylated IκB recruits the E3 ligase SCF-βTRCP, followed by its degradation in the proteasome Orian et al. After IκB degradation, NF-κB was then free to migrate to the nucleus of the cell, and induce transcription of target genes, including Beclin1 and p62, and induce autophagy Copetti et al.

Another level of regulation involved TNF-α receptor-associated protein complexes. Ubiquitylated RIPK1 could recruit NEMO and TAB-TAK1 complex for IKK activation and hence NF-κB stimulation.

Additionally, RIPK1 could also be modified by A20 through addition of Klinked poly-ubiquitin chains, sending the kinase for proteasomal degradation Kravtsova-ivantsiv et al. However, in some contexts, TNF-α-induced NF-κB activation was reported to inhibit autophagy Djavaheri-Mergny et al.

Furthermore in some contexts, RIPK1 silencing activated autophagy under both basal and stress conditions Yonekawa et al. On the other hand, RIPK1 itself was reported to be a target of pmediated selective autophagy Goodall et al. Moreover, autophagy was responsible for the degradation of NF-κB activator NIK and IKK complex subunits, indicating the presence of a tight cross-regulation of the NF-κB pathway by the UPS and autophagy Qing et al.

Another transcription factor that was controlling the autophagic outcome was HIF1α. Hypoxia induced HIF1α transcriptionally regulated various hypoxia response genes, including GLUT1 Chen et al.

HIF1α itself was regulated in a UPS-dependent manner. Under normoxia, hydroxylation of HIF1α specific prolyl hydroxylases PHDs hydroxylated HIF1α Jaakkola et al. In contrast, during hypoxia, PHDs were inhibited and HIF1α stabilized.

SCF E3 ligase complex was also a regulator of HIF1α stability in response to GSK3β-mediated phosphorylation of the protein Cassavaugh et al. Another E3 ligase facilitating HIF1α degradation was HAF also known as SART1 Unlike pVHL, HAF-mediated ubiquitylation of HIF1α was not depending on the oxygen levels, providing an alternative HIF1α regulation mechanism Koh et al.

Stability of PHD proteins were also controlled by the UPS. Moreover several DUBs were implicated in HIF1α regulation, including USP20 Li et al. FOXO family of transcription factors FOXOs were associated with various cellular pathways, including autophagy Zhao et al.

The activity of FOXOs were regulated by their phosphorylation status and following activation, FOXOs translocated to the nucleus and triggered the expression of a number of genes associated with different stages of the autophagy pathway, including ATG4 , ATG12 , BECN1 , ULK1 , PIK3C3 , MAP1LC3, and GABARAP Mammucari et al.

There are several connections between FOXOs and autophagy. Activation of the AKT pathway inhibited FOXO3 activity, led to a decrease in LC3 and BNIP3 expression, therefore blocked autophagy Stitt et al. On the other hand, AMPK activation led to the phosphorylation of FOXO3a and ULK1, inducing MAP1LC3 , GABARAP, and BECN1 expression and subsequent autophagy activation Sanchez et al.

Moreover, JNK deficiency in neurons increased autophagic activity through FOXO1-mediated BNIP3 upregulation and Beclin1 disassociation from BCL-XL Xu et al. Another example of a link between FOXOs autophagy involved ATG Liver specific knockout of FOXOs resulted in the downregulation of ATG14 and this event was associated with high levels of triglycerides in the liver and serum of mice Xiong et al.

Additionally, GATA-1 shown to directly regulate FOXO3-mediated activation of LC3 genes to facilitate autophagic activity Kang et al. Phosphorylation of FOXO proteins by various protein kinases, including AKT, IKK, and ERK, affected their ubiquitylation by E3 ligases and their stability Huang and Tindall, For instance, AKT-mediated phosphorylation of FOXO1 provided a signal for its recognition by the SKP protein, an SCF E3 ligase complex component, followed by FOXO1 ubiquitylation and degradation Huang et al.

COP1 was also identified as an E3 ligase that regulated FOXO protein stability. COP1 ubiquitylated FOXO1 and promoted its proteasomal degradation.

This type of regulation might be important in the glucose metabolism of hepatocytes, and possibly in autophagy modulation under this conditions Kato et al. Another FOXO regulating E3 ligase was MDM2 that was reported to be responsible for FOXO1 and FOXO3A ubiquitylation and degradation Fu et al. MDM2-mediated ubiquitylation was activated by the phosphorylation of FOXOs by AKT.

Due to its role in p53 regulation, MDM2 could be part of a more complex regulatory mechanism which might link the UPS, transcriptional regulation and autophagic activity. NRF2-KEAP1-P62 pathway was defined as another major oxidative stress response mechanism involving an interplay between the UPS and autophagy.

NRF2 is a transcription factor, and when activated, is upregulated antioxidant and metabolic enzymes, including TXNRD1 Suvorova et al. KEAP1 is an adaptor protein of the E3 ligase Cullin-3 and plays a role in substrate recognition.

Under normal conditions, transcription factor NRF2 was found in association with KEAP1-Cullin-3 E3 ligase complex, that catalyzed its ubiquitylation, rendering it a substrate for proteasomal elimination by selective autophagy Ishimura et al.

Competition resulted in the migration of free NRF2 to the nucleus and transactivation of stress-related cytoprotective genes Kobayashi et al. Additionally, the NRF2—KEAP1 pathway provides a positive feedback loop for autophagy. P62 was characterized as a direct transcriptional target of activated NRF2 Jain et al.

Moreover, KEAP1 regulation by p62 was modulated by the E3 ligase TRIM NRF2 activation was negatively affected by TRIMmediated Klinked ubiquitylation of p62 Pan et al. Crosstalk between autophagy and the UPS may change character under disease conditions, contribute to the pathogenesis of diseases and even affect their outcome.

Degenerative diseases and cancer are examples of diseases that illustrate the interplay between the UPS and autophagy in the clearance of misfolded abnormal proteins Juenemann et al.

For example, Huntington Disease is caused by poly-glutamine extensions in a protein called Huntingtin Htt , leading to abnormal organization and eventual aggregation of the protein. Htt protein was shown to be ubiquitylated via K or Klinked ubiquitin chains Bhat et al.

Mutant Htt clearance depended on both the UPS and autophagy in different experimental settings. Mutant Htt aggregates were largely cleared by Kdependent autophagy mechanisms Renna et al.

On the other hand, overexpression of Kspecific E3 ligase Ube3a, resulted in a UPS-dependent degradation of mutant proteins. Yet, cellular levels of E3 ligase was shown to decline in an age-dependent manner. Therefore, in elderly people, accumulation of Klinked polyubiquitylated proteins might tip the balance toward clearance of protein aggregates by autophagy.

A similar UPS switch was also observed in a CHIP-dependent manner Jana et al. Yet, autophagy was still functional under these conditions, and could significantly eliminate these aggregates Bayraktar et al. Therefore, preferential elimination of mutant proteins by autophagy might tip the balance in favor of wild-type proteins and restore disease-related loss of cellular functions including UPS-related mechanisms.

The role of the crosstalk between the two systems is also prominent in the cancer context. For example, the Pregulated and cancer-related protein EI24, was introduced as a critical link between the UPS and autophagy Devkota et al.

EI24 controlled the stability of E3 ligases TRIM41, TRIM2, and TRIM28 by the regulation of their autophagic degradation Devkota et al. Cellular levels of other E3 ligases, namely MDM2 and TRAF2, were also regulated by EIcontrolled degradation, modulating p53 and mTOR pathways, respectively, and influencing cancer formation and progression Devkota et al.

Changes include, modulation of levels of E3 ligases such as MDM2 Haupt et al. Under these circumstances, dynamic and complex changes in the regulation of the degradative pathways should have dramatic effects that contribute to cancer-related alterations in the proteomic landscape of cells.

Autophagy-UPS crosstalk emerges as a critical factor that determines the success of disease treatment, chemotherapy is one striking example. For instance, proteasome inhibition by the chemotherapy agent bortezomib resulted in the accumulation of misfolded proteins and induced compensatory autophagy in cancer cells Obeng et al.

Under these circumstances, autophagic activity protected cancer cells from bortezomib-induced cell death, and inhibition of autophagy improved the outcome of chemotherapy. These dual autophagy-UPS targeting approaches also gave promising results in clinical trials Vogl et al.

Several companies are now developing drugs that modulate the UPS or autophagy [for example, Huang and Dixit, ]. Concepts and data that were discussed above and elsewhere indicate that, depending on the disease type and treatment strategy, the crosstalk between the UPS and autophagy should definitely be taken into account in these efforts.

Autophagy and the ubiquitin proteasome systems are major degradation systems in mammalian cells that allow recycling of cellular contents ranging from soluble proteins to intracellular organelles.

Although their mode of action and their requirements for substrate recognition are different, there are several overlaps and interconnections between the UPS and autophagy pathways. A prominent component of the crosstalk is the ubiquitin protein itself and ubiquitylation.

Indeed, ubiquitin is a common signal for both the UPS and autophagy. It was proposed that, ubiquitin chain type could determine the pathway of choice for protein degradation.

Klinked ubiquitylation was introduced to be a signal for the UPS, whereas Klinked ubiquitylation directed proteins for autophagosomal degradation Herhaus and Dikic, Yet, a number of independent studies provided evidence that both ubiquitylation types could lead to autophagic degradation of substrates Wandel et al.

Moreover, recent studies underline the importance of ubiquitin phosphorylation as an event that increased the affinity of autophagy receptors for their targets during selective autophagy Kane et al. Additionally, non-ubiquitin modifications e. were shown to affect protein degradation as well Hwang and Lee, Therefore, a barcode of ubiquitin and other modifications seem to prime proteins for one or the other degradation pathway and determine their fate.

As another level of regulation, deconjugating enzymes such as DUBs may counteract or redirect proteins for different degradation systems. E3 ligases emerged as important components of the UPS-autophagy switches.

For example, Cullin-3 Pintard et al. On the other hand, the same E3 ligase that might be able to generate different ubiquitin linkages under different conditions and on different substrates Chan et al. A prominent example is the Parkin protein. During mitophagy although some of the proteins that are ubiquitylated by Parkin are degraded, other ubiquitylated proteins contribute to mitochondrial clustering and recognition by autophagy receptors.

To date, factors or modifications that determine the substrate selectivity of Parkin are unknown. Signaling switches involved in the regulated activation of one or the other system was shown to modify cellular responses to stress.

For example, NRF2 degradation by the UPS was controlled through pmediated KEAP1 elimination by autophagy Jain et al. Prevention of HIF1α degradation by the UPS, resulted in the expression of stress response genes, including autophagy genes, led to autophagy activation.

In another example, the UPS activity was required for NF-κB activation and NF-κB-mediated autophagy gene upregulation. Yet, autophagic degradation of NF-κB activators NIK and IKKs provided a negative feedback loop in the control in this context Qing et al. Degradation of the components or regulators of one system by the other system was also reported.

For example, proteasomes were defined as substrates of selective autophagy Marshall et al. Conversely, various autophagy proteins were ubiquitylated and degraded by the UPS in a regulated manner. Therefore, checks and balances between the two systems exist, and these control mechanisms possibly allow remodeling of the cellular proteome under different conditions.

Compensation mechanisms are also operational between the two systems. Inhibition of the UPS generally upregulated autophagy, whereas failures in the autophagy system were associated with increased UPS activity, although inefficient compensation and failure in both systems were also observed under certain conditions Korolchuk et al.

Moreover, alternative protein degradation pathways, such as CMA and microautophagy might come into play under these conditions as well.

Nevertheless, depending on the character of the target to be degraded, compensation mechanisms were less or more effective. For example, large aggregates and whole organelles should be cleared by autophagy, but defective ribosomal products that could not be accumulated in stress granules were shown to be directed for proteasomal degradation.

Therefore for cellular homeostasis and for proper functioning of cells, ideally both systems should be fully operational. Data obtained so far demonstrate that crosstalk and communication between autophagy and the UPS generally rely on non-specialized and even indirect links.

Yet, there might exist so far unknown specialized proteins providing coordination and co-regulation of the two systems. Furthermore, regulation through direct protein-protein interactions between known system components is another possibility. Therefore, dedicated communication proteins or pathways between the degradation mechanisms may be present, allowing better and faster coordination in case of need.

Further studies are required to unveil the nature of these putative proteins, interactions and pathways. An emerging theme in the regulation and coordination of autophagy and the UPS involves non-coding RNAs and their intricate networks. A growing list of microRNAs as well as long non-coding RNAs were implicated in the control of autophagy Tekirdag et al.

MicroRNAs have the advantage of affecting the level of multiple proteins at once, and they are able to rapidly reshape cellular signaling mechanisms and pathways. Therefore, non-coding RNA networks possibly contribute to the co-regulation of these degradative systems.

Intriguingly, deregulation of non-coding RNA levels contribute to the progression of diseases such as cancer. Future studies on non-coding RNAs will reveal their relevance in the autophagy-UPS crosstalk under physiological and pathological conditions.

Overall, coordination, interconnection and crosstalk mechanisms between the UPS and autophagy exist at various levels. In addition to ubiquitin and ubiquitylation, several proteins and signaling pathways were implicated in the communication and mutual regulation of the two systems.

Considering the importance of protein catabolism for cellular and organismal homeostasis and health, a better understanding of individual systems as well as the interconnections and crosstalks between them will be most rewarding from both a basic science perspective and with regards to clinical management of diseases involving protein quality control problems.

NK and DG wrote the manuscript and did critical reading. NK prepared the illustrations in the manuscript. This work was supported by the Scientific and Technological Research Council of Turkey TÜBİTAK Grant Project Number T and Sabanci University. NK was supported by TUBITAK-BIDEB A Ph.

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Jin, S. USP19 modulates autophagy and antiviral immune responses by deubiquitinating Beclin Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL.

Johnson, J. Exome sequencing reveals VCP mutations as a cause of familial ALS.

In another study, proteasome inhibition was associated with an increase in p62 and GABARAPL1 levels by Nrf1-dependent and -independent pathways prior to autophagy activation Sha et al. Autophagy induction following proteasome inhibition correlated with AMPK activation as well.

A number of studies provided evidence that proteasomal inhibition is sensed by both AMPK and mTORC1, two major regulators of autophagy. For instance, in macrophages, epitelial and endothelial cells, proteasome inhibition using chemicals resulted in the activation of AMPK Xu et al.

In some other cancer cell types, CaMKKβ and glycogen synthase kinase-3β GSK-3β were identified as upstream regulators of AMPK activation, proteasome inhibition was linked to a decrease in GSK-3β activity and to the activation of AMPK and autophagy Sun et al.

On the other hand, Torin or rapamycin-mediated inhibition of mTOR stimulated long-lived protein degradation through activation of both UPS and autophagy Zhao et al.

In retinal pigment epithelial cells, inhibition of proteasome by lactacystin and epoxomicin was shown to block the AKT-mTOR pathway and induce autophagy Tang et al.

SiRNA-mediated knockdown of Psmb7 gene coding for the proteasome β2 subunit, resulted in enhanced autophagic activity, and it was linked the mTOR activation status of cultured cardiomyocytes Kyrychenko et al.

Similarly, impairment of autophagy correlated with the activation of the UPS. In colon cancer cells, chemical inhibition of autophagy and small RNA mediated knock down of ATG genes resulted in the upregulation of proteasomal subunit levels, including the catalytic proteasome β5 subunit, PSMB5 and led to increased UPS activity Wang et al.

In another study, 3-MA-mediated autophagy inhibition in cultured neonatal rat ventricular myocytes NRVMs increased chymotrypsin-like activity of proteasomes Tannous et al.

Since proteasomes were identified as autophagic degradation targets proteaphagy , enhanced proteasome peptidase activity following autophagy inhibition might be associated with the accumulation of proteasomes Cuervo et al.

Yet in several cases, autophagy inhibition correlated with the accumulation of ubiquitylated proteins. For instance in independent studies with ATG5 or ATG7 knockout mice, accumulation of ubiquitylated conjugates were observed, especially in the brain and the liver of the animals Komatsu et al.

Similar results were observed in other animal models such as Drosophila Nezis et al. In line with these data, inhibition of autophagy through siRNA-mediated knockdown of ATG7 and ATG12 in HeLa cells resulted in the impairment of UPS, accumulation of ubiquitylated proteins as well as other important UPS substrates, including p53 and β-catenine Korolchuk et al.

In above-cited papers, autophagy impairment followed by the autophagy receptor p62 accumulation in cells, and played a key role in the observed UPS defects. Ubiquitylation was proposed to be a common component that directs substrates to the proper degradation system and even contribute to the UPS-autophagy crosstalk Korolchuk et al.

According to this view, proteins that are predominantly linked to Kbased ubiquitin chains are generally directed for degradation through UPS.

Conversely, aggregates that are linked to Kbased ubiquitin chains are directed for autophagic degradation. P62 binding capacity was introduced as the critical step in the choice between the UPS and autophagy. Although, p62 is able to attach both K and Klinked ubiquitin chains through its UBA domain, binding affinity of the protein for Klinked chains seems to be higher Long et al.

Due to this dual ubiquitin binding ability, p62 might show UPS inhibitory effects in some contexts. In summary, in the case of a defect in one of the two degradation systems, the other system is upregulated in order to eliminate ubiquitylated protein substrates.

Yet, compensation does not always work and its success largely depends on cell types, cellular and environmental conditions and target protein load. Function of proteins depend on their proper folding and 3D structures. Various insults, including heat shock, organellar stress, oxidative stress etc.

Moreover several disease-related mutations were associated with folding problems. Failure to refold result in dysfunctional or malfunctional, hence toxic protein accumulations, activation of stress and even cell death pathways.

In order to control toxic protein accumulations, an active process of protein aggregate formation comes into play.

Additionally some proteins, including mutant proteins are already prone to form aggregates. Selective clearance of most cytosolic proteins require ubiquitylation.

Depending on their solubility, ubiquitylated proteins and protein aggregates are then cleared by the UPS or autophagy. Soluble fractions of proteins with a folding problem are recognized by the chaperone machinery and directed to the UPS for degradation.

BAG family proteins, especially BAG1, interact with the Hsp70 complex and induce proteasomal degradation of client proteins. On the other hand, clearance of insoluble aggregate-prone proteins require formation of aggresomes.

Ubiquitylation by a number of different E3 ligases, including CHIP, Parkin, HRD1 and TRIM50 prime aggregate-prone proteins Olzmann et al. HDAC6 is another protein that plays a key role in the process of aggresome formation. HDAC6 was shown to provide the link between Kbased ubiquitylated aggregates and microtubule motor protein dynein Matthias et al.

Then, dynein-mediated mechanism direct the aggregates toward microtubule organizing centers MTOCs , resulting in their piling of as aggresomes Johnston et al.

Following aggresome formation, direct interaction of adaptor proteins p62 and NBR1 with ubiquitylated aggregates result in their delivery to autophagosomes Ichimura et al. Another autophagy-related protein, ALFY, was also identified as a player in the selective autophagy and degradation of aggresomes Clausen et al.

FIGURE 4. Misfolded proteins can be eliminated by both the UPS and autophagy system. Misfolded proteins are ubiquitylated and based on the differences in ubiquitin linkages and ubiquitin binding proteins, they are directed for proteasomal degradation or further accumulated in aggresomes.

Aggresomes are selectively cleared by autophagy. An alternative pathway for aggresome formation require Hsp70 partner proteins BAG3 and CHIP Zhang and Qian, Similar to HDAC6, BAG3 binds to dynein, and this directs Hsp70 substrates to aggresomes. However, BAG3-dependent aggresome formation was not dependent on the ubiquitylation of substrates as in the case of HDAC6, and CHIP E3 ligase activity was dispensible Gamerdinger et al.

Yet, E3 ligases such as CHIP were required for BAG3-dependent aggresome clearance by autophagy Klimek et al. Until so far, we focused on the UPS and autophagy as complementary but independent mechanisms. However, there are cases where components of one system were reported to be a proteolytic target of the other system.

For example, a number of autophagy proteins were regulated through degradation by the UPS. On the other hand, even the whole proteasomes were shown be selective targets of autophagic degradation. Here, we will give examples of how mutual regulation through proteolysis contributes to the crosstalk and the interplay between the two systems.

Early studies indicated that proteasomes could be degraded in lysosomes Cuervo et al. Later on, plant studies revealed that lysosomal degradation of 26S proteasomes occurred by a specific form of selective autophagy, proteaphagy Marshall et al.

RPN10 protein was introduced as an ATG8 interacting plant proteaphagy receptor. Instead, Cue5 protein in the yeast and its human ortholog TOLLIP, were introduced as selective receptors regulating proteasome clearance by autophagy Lu et al. Moreover, p62 was also described as another proteaphagy receptor Cohen-kaplan et al.

For example, in mammals, amino acid starvation significantly upregulated ubiquitylation of 19S proteasome cap components RPN1, RPN10, RPN13, and led to their pmediated recruitment to autophagosomes Cohen-kaplan et al.

Interestingly during carbon or nitrogen starvation, plant and yeast proteasomes were shown to localize in proteasomal storage granules PSGs , protecting them from autophagic degradation during stress Peters et al. Whether similar mechanisms exist in the mammals is currently an open question.

These observations underline the importance of selective degradation of proteasome by autophagy in the control of proteasome numbers as well as overall UPS and lytic activity in cells.

FIGURE 5. Schematic representation of the selective degradation of proteasomes by autophagy. Upon starvation and functional defects proteasomes become ubiquitylated and degraded by autophagic machinery. Modulation of the half-life of some proteins in the autophagy pathway by the UPS serves as a means to control cellular autophagic activity.

For instance, LC3 protein was shown to be processed in a stepwise manner by the 20S proteasome, a process that was inhibited by p62 binding Gao et al. On the other hand, E3 ligase NEDD4-mediated Klinked ubiquitylation of Beclin1 prevented its binding to the lipid kinase VPS34, and led to its degradation Platta et al.

Another E3 ligase, RNF ubiquitylated Beclin1 adding Klinked ubiquitin chains on the protein Xu et al. Beclin1 ubiquitylation resulted in autophagy blockage in both cases.

Conversely, reversal of Beclin1 ubiquitylation by the DUB protein USP19 stabilized the protein under starvation conditions and promoted autophagy Jin et al.

USP10 and USP13 as well as USP9X were characterized as other DUBs that regulated autophagy through control of Beclin1 stability Liu et al.

Beclin1 is not the only autophagy protein that is targeted by the UPS in a controlled manner. G-protein-coupled receptor GPCR ligands and agonists were reported to regulate cellular Atg14L levels, and therefore autophagy, through ZBTBmediated ubiquitylation of the protein Zhang T.

et al. Serum starvation increased GSK3β-mediated phosphorylation of ZBTB16, leading to its degradation. Under these conditions, stabilization of Atg14L restored of autophagy. AMBRA1 is another UPS-controlled autophagy protein.

Cullin-4 was identified as an E3 ligase that was responsible for the ubiquitylation of AMBRA1, dooming it for degradation under nutrient-rich conditions where autophagy should be inhibited Antonioli et al. The PI3K complex subunit p85b is another example. Ubiquitylation of this autophagy signaling component by the E3 ligase SKP1 led to a decrease in its cellular levels and stimulated autophagic activity Kuchay et al.

Ubiquitylation of some autophagy proteins did not result in their immediate proteasomal degradation, yet the post-translational modification provided an extra layer of control for the autophagy pathway.

For instance, autophagy receptor OPTN was ubiquitylated as a target of the E3 ligase HACE1, and Klinked ubiquitylation regulated the interaction of the protein with p62 Liu Z. TRAF6, a central E3 ligase of the NF-κB pathway, participated controlled ULK1 activity through Klinked ubiquitylation.

Under nutrient-rich conditions, mTOR phosphorylated AMBRA1 leading to its inactivation. When nutrients were limiting, mTOR inhibition resulted in AMBRA1 dephosphorylation and increased the interaction of the protein with TRAF6.

This event facilitated ULK1 ubiquitylation by TRAF6 Nazio et al. Ubiquitylation of ULK1 resulted in the stabilization of the protein, controlled its dimerization and regulated its kinase activity.

Another ubiquitin-dependent regulation mechanism involved AMBRA1-Cullin-5 interaction in the regulation of mTOR complex component DEPTOR Antonioli et al.

Above-mentioned AMBRA1-Cullin-4 complex dissociated under autophagy-inducing conditions, allowing AMBRA1 to bind another E3 ligase, Cullin This newly formed complex was shown to stabilize DEPTOR and induce mTOR inactivation, providing a negative feed-back loop in the control of autophagy Antonioli et al.

In another study, TLR4 signaling triggered autophagy through Beclin1 ubiquitylation and stabilization. TLR4-associated TRAF6 protein was identified as the E3 ligase responsible for Klinked ubiquitylation of Beclin1 at its BH3 domain.

This modification blocked inhibitory BCL-2 binding to the protein, and free Beclin1 could activate autophagy Shi and Kehrl, On the other hand, the deubiquitinating enzyme A20 reversed TRAF6-mediated ubiquitylation of Beclin1, resulting in autophagy inhibition Shi and Kehrl, Another Klinked ubiquitylation event on Beclin1 was promoted by AMBRA1 protein.

In the same context, the WASH protein interacted with Beclin1, blocked AMBRA1-mediated Beclin1 ubiquitylation, and suppressed autophagy Xia et al. LC3 and p62 were also subjected to regulatory ubiquitylation. NEDD4 was identified as the E3 ligase in these reactions.

NEDD4 was reported to interact with LC3 Sun et al. Moreover, NEDD4 deficient cells exhibited aberrant p62 containing inclusions, indicating the defect in aggresome clearance Lin et al.

Hence, NEDD4 is important for the regulation of p62 function and autophagy. Another essential function of autophagy is the clearance of intracellular pathogens. This special form of autophagy, called xenophagy, is a result of a cooperation between the ubiquitylation machinery and the autophagy pathway.

Pathogens such as Streptococcus pyogenes, Mycobacterium tuberculosis, Listeria monocytogenes, and Shigella flexneri were identified as autophagy targets Gutierrez et al. As a form of selective autophagy, xenophagy involves cargo labeling with ubiquitin, followed by the recognition by autophagy receptors Figure 6.

K and Klinked and linear M1-linked ubiquitin chains were shown to mediate recognition of different pathogens by the xenophagy machinery Collins et al.

FIGURE 6. Selective degradation of invaders by xenophagy is example of coregulation of the UPS and autophagy. Cellular degradation of invading bacterium was ubiquilated by various E3 ligases and recognized by adaptor proteins for recruitment autophagic membranes around bacterium.

For example, Salmonella enterica serovar Typhimurium was heavily ubiquitylated in mammalian cells, and activation of xenophagy restricted intracellular bacteria numbers Birmingham et al. Recent studies showed that, bacterial outer membrane-associated and integral membrane proteins were targets of ubiquitylation Fiskin et al.

A number of E3 ligases were involved in xenophagy, including Parkin, RNF, ARIH1, HOIP, and LRSAM1 Huett et al. For example, both K and Klinked ubiquitylation were observed on Mycobacterium, and Parkin was identified as the E3 ligase catalyzing the Klinked ubiquitylation Collins et al.

Moreover endosome-free areas on the intracellular Salmonella Typhimurium contained a directly attached ubiquitin coat, and addition of linear M1-linked ubiquitin chains by the E3 ligase HOIP of the LUBAC on these ubiquitins contributed to the autophagy of the intracellular parasite Noad et al.

Xenophagy receptors that were described to date include p62, OPTN, NDP52, and NBR1 Thurston, ; Zheng et al. The interplay between ubiquitylation and autophagy achieves the important task of keeping host cells pathogen-free and providing an intracellular innate immune defense mechanism against invaders.

In some reports, ubiquitylated bacteria were found to be surrounded by proteasomes as well Perrin et al. Whether in the elimination of invading organisms, the crosstalk between the UPS and autophagy systems goes beyond ubiquitylation, needs further consideration.

As discussed below, cellular mechanisms controlling commensal-turned ancient intracellular microorganisms, namely mitochondria, indeed rely on the function of both the UPS and autophagy.

Mitochondria are vital organelles that form an intracellular dynamic network in the cytosol of eukaryotic cells. Through fusion and fission, they are constantly made and destroyed. Under steady state conditions, mitochondria might be eliminated by basal in a non-selective manner. On the other hand, elimination of damaged, dysfunctional or superfluous mitochondria requires a selective form of autophagy called mitophagy Lemasters, Programmed elimination of mitochondria during development and differentiation e.

Recent studies showed that mitophagy is a biological phenomenon that involves both the UPS and autophagy. In this section, we will discuss mechanisms of mitophagy, and analyze connections between the UPS and autophagy in this context.

Depending on the E3 ligase that ubiquitylates proteins on mitochondria, mitophagy can be divided into two major forms: Parkin-dependent and Parkin-independent mitophagy. Strikingly, Parkin recruitment to mitochondria was found to be necessary for mitophagy Narendra et al.

FIGURE 7. Mitochondrial elimination by autophagy requires the activity of both the UPS and autophagy. Under normal conditions, after being synthesized as precursor in the cytoplasm, PINK1 was imported to mitochondria by its N-terminal mitochondria targeting sequence MTS. Then, PINK1 was post-translationally modified within mitochondria by resident proteases: MPP and PARL Jin et al.

Cleavage by PARL resulted in destabilization of the protein and its degradation by cytoplasmic proteasomes Yamano and Youle, Under mitochondrial stress however, PINK1 cleavage did not occur and the protein accumulated on the outer mitochondrial membrane OMM Lazarou et al.

Recruitment of cytoplasmic E3 ligase Parkin onto mitochondria required stabilization and the kinase activity of the PINK1 protein Lazarou et al. Parkin itself was a substrate of PINK1 Kondapalli et al.

Phosphorylation of Parkin by PINK1 resulted in a conformational change overcoming an autoinhibition, and stimulated its E3 ligase activity Kondapalli et al. Interestingly, PINK1 was shown to phosphorylate ubiquitin molecules on mitochondrial resident proteins as well.

Ubiquitin phosphorylation correlated with an increase in the amount of mitochondria-localized Parkin, providing a feed-forward mechanism of Parkin recruitment Kane et al.

Several proteins on the mitochondrial outer membrane were identified as Parkin ubiquitylation substrates. The list includes VDAC, TOM proteins, mitofusins etc Sarraf et al.

Following ubiquitylation some of these targets were shown to be degraded by the proteasome e. Degradation of proteins related to mitochondrial integrity promoted fission events that facilitate engulfment of mitochondrial portions by autophagosomes, whereas proteins that are not degraded upon ubiquitylation rather contributed to mitochondrial rearrangements e.

The UPS activity was a prerequisite in the preparation of mitochondria for autophagy. Ubiquitylation of mitochondrial targets preceeded the recruitment of the autophagic machinery onto mitochondria Yoshii et al. Serial knock out of putative autophagy receptors showed that NDP52, optineurin OPTN and TAX1BP1 were functional mitophagy receptors, and a triple knockout of these proteins completely blocked mitophagy Lazarou et al.

On the other hand, the autophagy receptor p62 was essential for clustering of damaged mitochondria in perinuclear region of the cells, but not for mitophagy Narendra et al. Ubiquitin modifications on mitochondria might be reversed by the action of DUB proteins.

Several DUBs were identified as positive or negative regulators of mitophagy Dikic and Bremm, ; Wang et al. For example, deubiquitylation of mitochondrial targets by USP15, USP30, and USP35 prevented further progression of mitophagy in a number of cell lines and experimental models Bingol et al.

DUB-mediated deubiquitylation of targets decreased Parkin recruitment onto mitochondria as well Bingol et al. USP8-mediated removal of K6-linked ubiquitin chains from Parkin itself affected recruitment of the protein onto mitochondria and therefore mitophagy Durcan et al.

Expression of Parkin is restricted to a few cell types, including dopaminergic neurons. Consequently, Parkin-null animals showed prominent mitophagy defects only in selected brain regions Lee et al. Therefore in other cell types and tissues, mitophagy has to proceed in a Parkin-independent manner.

Alternative E3 ligases were found to play a role in mitophagy in these contexts. Mulan MUL1 is an E3 ubiquitin ligase that resided on the OMM, and it was shown to play a role in Parkin-independent mitophagy in different model organisms, including Caenorhabditis elegans , Drosophila and mammals Ambivero et al.

Mulan stabilized DRP1, led to degradation of MFN2, and interacted with ATG8 family member protein GABARAP Braschi et al.

Another E3 ligase that was associated with mitophagy was GP78 Christianson et al. Over expression of GP78 induced MFN1 and 2 ubiquitylation and degradation, that was followed by mitochondrial fragmentation and mitophagy in cells lacking Parkin Fu et al. Synphilindependent recruitment of the E3 ligase Siah1 to mitochondria resulted in mitochondrial protein ubiquitylation and mitophagy in a PINK1-dependent but Parkin-independent manner Szargel et al.

Conversely, another OMM E3 ligase, MITOL MARCH5 , was reported to ubiquitylate FIS1, DRP1 Yonashiro et al. All these findings underline the fact that mitophagy might proceed in cells which do not express Parkin.

Further studies are required to unravel the molecular mechanisms of Parkin-independent mitophagy in different tissues and cell types, and reveal the details of the crosstalk between the UPS and autophagy under these conditions. During differentiation, in order to increase their capacity to load hemoglobin-bound oxygen, reticulocytes lose their organelles, including mitochondria, and become mature red blood cells Dzierzak and Philipsen, During this process, a protein called NIX also known as BNIP3L is upregulated Aerbajinai et al.

NIX is a C-terminally anchored outer mitochondrial membrane OMM protein that contains a LC3-interacting region LIR at its cytoplasmic N-terminal part.

Through its LIR domain, NIX interacted with LC3, enabling engulfment of mitochondria by autophagosomes in reticulocytes Novak et al. Characterization of NIX-deficient mice showed that, NIX-deficient Erythrocytes failed to eliminate their mitochondria revealing a critical role for NIX in mitophagy Schweers et al.

Although NIX-dependent mitophagy was predominantly studied in reticulocytes, NIX-dependent mitophagy might be important for other cell types as well [for example, see Esteban-Martínez et al. Autophagy of peroxisomes, pexophagy, is a selective degradation process of peroxisomes during which the UPS and autophagy mechanisms work in collaboration.

Peroxisomes are responsible of a number of cellular functions, including fatty acid oxidation, purine metabolism and phospholipid synthesis Wanders et al. Several peroxisomal enzymes are involved in redox regulation due to their dual functions in the generation and scavenging of reactive oxygen and nitrogen species.

Therefore, peroxisome biogenesis and degradation must be tightly regulated in order to control peroxisome size, number and function Du et al. Moreover under stress conditions such as hypoxia, oxidative stress, starvation or conditions causing UPS defects, pexophagy is upregulated.

During pexophagy, a number of peroxisomal membrane proteins, including peroxins and PMP70 become ubiquitylated Kim et al. PEX2-PEXPEX12 complex serves as an E3 ligase at least for two well studied peroxisome proteins, PEX5 and PMP For example, PEX2 overexpression or amino acid starvation activated the ubiquitylation of PEX5, and another peroxisomal membrane protein, PMP70, and led to peroxisome degradation Sargent et al.

Moreover in response to oxidative stress, ATM was recruited onto peroxisomes through physical interaction with PEX5 and promote its ubiquitylation. Inactivation of mTORC1 in a TSC2-dependent manner and stimulation of ULK1 phosphorylation by ATM, potentiated pexophagy Zhang J.

On the other hand, AAA ATPase complex PEX1, PEX6, and PEX26 was shown to extract ubiquitylated PEX5 from peroxisomal membranes and regulate pexophagy Carvalho et al. Both NBR1 and p62 were shown to be recruited onto peroxisomes during pexophagy.

Yet, NBR1 was a major pexophagy receptor in a number of contexts, and p62 increased the efficiency of NBR1-dependent pexophagy through direct interaction with the latter Deosaran et al.

Altogether, these findings underline the importance of ubiquitylation for the selective degradation of peroxisomes by autophagy.

FIGURE 8. Selective removal of peroxisomes by autophagy utilizes ubiquitylation as signal. In addition to major cellular organelles, autophagy was implicated in the clearance of ribosomes. Although ribosomes can be degraded in a non-specific manner during non-selective autophagy, a special form of selective autophagy is activated under various stress conditions, and the process is called ribosomal autophagy or ribophagy.

On the other hand, mRNA protein complexes that are stalled during translation form stress granules, and their clearance requires both the UPS and autophagy.

In the mammalian system, in addition to mTOR inhibition, oxidative stress, induction of chromosomal mis-segregation, translation inhibition and stress granule formation were all shown to induce ribophagy An and Harper, Ubiquitylation of ribosomes was observed under ER stress-inducing conditions Higgins et al.

Yet, individual ribosomal proteins were indeed shown to be a target of the UPS Wyant et al. NUFIP1-ZNHIT3 proteins were identified as novel ribophagy receptors that directly connected ribosomes to LC3 and autophagy, yet whether ubiquitylation is a prerequisite for ribophagy needs to be clarified by future studies Wyant et al.

FIGURE 9. Ubiquitylation primes ribosomes and stress granules for proteasomal degradation and autophagic elimination. Stress granules are composed of actively accumulated non-translating mRNA ribonucleoprotein complexes Protter and Parker, Proteins that accumulated in the stress granules, include stalled 40S ribosomal units and various translation initiation factors [e.

G3BP1 and TIA-1 are also among the proteins that contribute to stress granule formation Kedersha et al. Moreover, an interplay between G3BP1 and Caprin1 proteins and the DUB protein USP10 was shown to regulate stress granule formation Kedersha et al. HDAC6 protein was a component of stress granules as well Seguin et al.

Endoplasmic reticulum ER stress is one of the conditions under which both the UPS and autophagy pathways are being activated. Abnormalities in calcium homeostasis, oxidative stress and conditions leading to protein glycosylation or folding defects etc.

ER stress might be very destructive for cells, therefore ER-specific stress response pathways such as the unfolded protein response UPR and the ER-associated degradation ERAD pathways were evolved.

Both pathways are directly or indirectly connected to the UPS and autophagy. In mammalian cells, accumulation of unfolded proteins in the lumen of the ER result in the activation of stress responses. PERK activation leads to the phosphorylation of the α subunit of the translation initiation factor, eIF2α, which inhibits the assembly of the 80S ribosome and cap-dependent protein synthesis, while allowing cap-independent translation of the stress response genes such as ATF4.

Activation of IRE1 and ATF6 promotes transcription of other stress response genes. IRE1-mediated processing generates a splice-form of the XBP1 mRNA, resulting in the production of a transcription factor that upregulates chaperones and other relevant genes.

Due to a decrease in the protein load in the ER and an increased folding capacity, the UPR facilitates recovery from stress. In case of failure, the UPR sensitizes cells to programmed death mechanisms.

FIGURE Crosstalk between the UPS and autophagy systems during ER stress and ERAD. Components of the UPR were subject to active regulation by the UPS. For example, SCF component E3 ligase βTrCP was shown to lead to the ubiquitylation ATF4 following its phosphorylation Lassot et al.

CHOP stability was regulated by the UPS and p and cIAP were responsible for CHOP ubiquitylation and degradation counterbalancing its upregulation during ER stress Qi and Xia, ; Jeong et al. Another UPR component, IRE1 was identified as a ubiquitylation target of the E3 ligase CHIP during ER stress.

Ubiquitylation IRE1 inhibited its phosphorylation, perturbed its interaction with TRAF2, and attenuating JNK signaling Zhu et al. Under stress conditions, translation of XIAP, an E3 ligase protein and an inhibitor of apoptosis was downregulated in a PERK-eIF2α-dependent manner. In the same context, ATF4 may promote ubiquitylation and degradation of XIAP, leading to sensitization of cells to ER stress-related cell death Hiramatsu et al.

Conversely, activation of PERK-eIF2α axis might also show opposing effects through induction of other IAP proteins, cIAP1 and cIAP2, and counter balance cell death induction signals Hamanaka et al.

Endoplasmic reticulum stress was shown to trigger autophagy, and ER-related stress response mechanisms were involved in the process. PERK-mediated phosphorylation of eIF2α and resulting ATF4 and CHOP activation, were associated with the transcription of genes such as ATG5, ATG12, Beclin1, ATG16L1, LC3, p62 and TSC2 activator, hence mTOR inhibitor REDD1 Whitney et al.

Moreover, CHOP downregulated BCL2 binding Mccullough et al. TRB3, an AKT inhibitor protein, was also described as a target of CHOP Ohoka et al.

In addition, IRE1 activation resulted in the recruitment of ASK1 by the adaptor TRAF2 and the outcome was the activation of JNK and p38 kinases Nishitoh et al. BCL2 is one of the targets of JNK, its phosphorylation by the kinase resulted in destabilization the inhibitory BCL2-Beclin1 complex, stimulating autophagy Bassik et al.

On the other hand, in its unspliced form, IRE1 splicing target XBP1, in its unspliced form was shown to target the autophagy activator FOXO1 for degradation by the UPS Vidal et al.

Endoplasmic reticulum is a major calcium store in cells, and calcium release to cytosol was observed during ER stress. In addition to problems with SERCA refill pumps and leakiness of membranes during stress, upregulation of ERO1-α by CHOP resulted in an IP3-mediated calcium release Li et al.

Calcium binding protein calmodulin senses the cytosolic increase in the concentration of the ion, and bind to calmodulin-regulated kinases such as CaMKII and DAPK1, modulating their activity.

Activated CaMKII was shown to stimulate autophagy through AMPK phosphorylation and activation Høyer-Hansen et al. In addition, calmodulin-binding and PP2A-mediated dephosphorylation was necessary for the activation of the autophagy-related kinase DAPK1 Gozuacik et al.

DAPK1 could directly phosphorylate Beclin1 on the BH3-domain, resulting in the dissociation of Beclin1 from the BCL2-Beclin1 complex and allowing it to stimulate autophagy Zalckvar et al. Proteins that accumulate in the ER are degraded by the ER-associated degradation ERAD system.

ERAD mediates transport, extraction and ubiquitylation of proteins that cannot be salvaged and target them for degradation in proteasomes. In mammalian cells, ER membrane-resident complexes containing E3 ligases such as HRD1 and GP78, and other regulatory components such as EDEM1, SEL1L, ERManI, and HERP control the ERAD pathway.

DUB proteins, including YOD1, USP13, USP19, and Ataxin-3 were implicated in the control of client protein ubiquitylation and ERAD substrate modulation Zhong and Pittman, ; Bernardi et al.

ER-associated degradation regulators and therefore ERAD might be controlled by the UPS and autophagy pathways. For example, E3 ligase Smurf1 was found to be downregulated during ER stress, resulting in the accumulation of its direct ubiquitylation target WFS, which is a stabilizer ER-related E3 ligase HRD1 Guo et al.

Smurf1 was also involved in selective bacterial autophagy Franco et al. On the other hand, while the ERAD complex component HERP protein was degraded by the UPS Hori et al. An ER-localized E3 ligase synoviolin protein was shown to ubiquitylate HERP protein and control its degradation by proteasome Maeda et al.

Yet, other ERAD-related components, EDEM1 and Derlin2 as well as ubiquitylated EDEM1 proteins colocalized with cytoplasmic aggregates and autophagy receptors p62 and NBR1, they were degraded by selective autophagy Le Fourn et al. ERManI, a mannosidase that is responsible for priming ER-resident glycosylated proteins for degradation, was described as an accelerator of the ERAD pathway and clearance of clients by the UPS.

But, following proteasome inhibition and subsequent ER stress, ERManI colocalized with LC3 and degraded in an autophagy-dependent manner Benyair et al. All these findings point out to the presence of important junctions and coregulation nodes between the UPS and autophagy in the context of ER stress.

Additionally, ERphagy, the autophagy of portions of the ER, was implicated in the recovery from ER stress and control of ER size, but this mechanism was so far described as a ubiquitin-independent process Schuck et al.

Several transcription factors that are regulated by the UPS, including p53, NFκB, HIF1α, and FOXO, have been implicated in the control of autophagy. In general, these factors were shown to directly activate transcription of key autophagy genes under stress conditions.

Some autophagy proteins such as LC3 are consumed in the lysosome following delivery, and during prolonged stress, cellular levels of these proteins are sustained by mechanisms, including transcription. On the other hand, regulation of the transcriptional activity NRF2 involves a special crosstalk between the two systems.

In this section, we will summarize molecular details of transcription regulation by the UPS and autophagy. P53, a guardian of the genome, is one of the well-known transcriptonal regulators that has a dual role in autophagy depending on its intracellular localization.

Accumulating p53 protein activates transcription of several stress- and death-related genes, including autophagy-related genes PRKAB1 , PRKAB2, TSC2 , ATG2, ATG4, ATG7 , ATG10 , ULK1 , BNIP3, DRAM1, and SESN2 Crighton et al.

On the other hand, a cytosolic form of p53 was shown to inhibit AMPK and activate the mTOR pathway. Additionally, another E3 ligase, NEDD was shown to control MDM2 stability and p53 activation Xu et al. In addition to MDM2, another E3 ligase, PIRH2, was able to ubiquitylate p53 to control its cellular stability Shloush et al.

NF-κB is a well studied transcriptional regulator of autophagy. As a result of its association with IκB, NF-κB is found in an inactive state in the cytosol. In response to agonists, IκB was reported to be ubiquitylated and subsequently degraded by the UPS.

Phosphorylated IκB recruits the E3 ligase SCF-βTRCP, followed by its degradation in the proteasome Orian et al. After IκB degradation, NF-κB was then free to migrate to the nucleus of the cell, and induce transcription of target genes, including Beclin1 and p62, and induce autophagy Copetti et al.

Another level of regulation involved TNF-α receptor-associated protein complexes. Ubiquitylated RIPK1 could recruit NEMO and TAB-TAK1 complex for IKK activation and hence NF-κB stimulation. Additionally, RIPK1 could also be modified by A20 through addition of Klinked poly-ubiquitin chains, sending the kinase for proteasomal degradation Kravtsova-ivantsiv et al.

However, in some contexts, TNF-α-induced NF-κB activation was reported to inhibit autophagy Djavaheri-Mergny et al. Furthermore in some contexts, RIPK1 silencing activated autophagy under both basal and stress conditions Yonekawa et al.

On the other hand, RIPK1 itself was reported to be a target of pmediated selective autophagy Goodall et al. Moreover, autophagy was responsible for the degradation of NF-κB activator NIK and IKK complex subunits, indicating the presence of a tight cross-regulation of the NF-κB pathway by the UPS and autophagy Qing et al.

Another transcription factor that was controlling the autophagic outcome was HIF1α. Hypoxia induced HIF1α transcriptionally regulated various hypoxia response genes, including GLUT1 Chen et al. HIF1α itself was regulated in a UPS-dependent manner.

Under normoxia, hydroxylation of HIF1α specific prolyl hydroxylases PHDs hydroxylated HIF1α Jaakkola et al. In contrast, during hypoxia, PHDs were inhibited and HIF1α stabilized. SCF E3 ligase complex was also a regulator of HIF1α stability in response to GSK3β-mediated phosphorylation of the protein Cassavaugh et al.

Another E3 ligase facilitating HIF1α degradation was HAF also known as SART1 Unlike pVHL, HAF-mediated ubiquitylation of HIF1α was not depending on the oxygen levels, providing an alternative HIF1α regulation mechanism Koh et al.

Stability of PHD proteins were also controlled by the UPS. Moreover several DUBs were implicated in HIF1α regulation, including USP20 Li et al. FOXO family of transcription factors FOXOs were associated with various cellular pathways, including autophagy Zhao et al.

The activity of FOXOs were regulated by their phosphorylation status and following activation, FOXOs translocated to the nucleus and triggered the expression of a number of genes associated with different stages of the autophagy pathway, including ATG4 , ATG12 , BECN1 , ULK1 , PIK3C3 , MAP1LC3, and GABARAP Mammucari et al.

There are several connections between FOXOs and autophagy. Activation of the AKT pathway inhibited FOXO3 activity, led to a decrease in LC3 and BNIP3 expression, therefore blocked autophagy Stitt et al. On the other hand, AMPK activation led to the phosphorylation of FOXO3a and ULK1, inducing MAP1LC3 , GABARAP, and BECN1 expression and subsequent autophagy activation Sanchez et al.

Moreover, JNK deficiency in neurons increased autophagic activity through FOXO1-mediated BNIP3 upregulation and Beclin1 disassociation from BCL-XL Xu et al.

Another example of a link between FOXOs autophagy involved ATG Liver specific knockout of FOXOs resulted in the downregulation of ATG14 and this event was associated with high levels of triglycerides in the liver and serum of mice Xiong et al. Additionally, GATA-1 shown to directly regulate FOXO3-mediated activation of LC3 genes to facilitate autophagic activity Kang et al.

Phosphorylation of FOXO proteins by various protein kinases, including AKT, IKK, and ERK, affected their ubiquitylation by E3 ligases and their stability Huang and Tindall, For instance, AKT-mediated phosphorylation of FOXO1 provided a signal for its recognition by the SKP protein, an SCF E3 ligase complex component, followed by FOXO1 ubiquitylation and degradation Huang et al.

COP1 was also identified as an E3 ligase that regulated FOXO protein stability. COP1 ubiquitylated FOXO1 and promoted its proteasomal degradation.

This type of regulation might be important in the glucose metabolism of hepatocytes, and possibly in autophagy modulation under this conditions Kato et al. Another FOXO regulating E3 ligase was MDM2 that was reported to be responsible for FOXO1 and FOXO3A ubiquitylation and degradation Fu et al.

MDM2-mediated ubiquitylation was activated by the phosphorylation of FOXOs by AKT. Due to its role in p53 regulation, MDM2 could be part of a more complex regulatory mechanism which might link the UPS, transcriptional regulation and autophagic activity. NRF2-KEAP1-P62 pathway was defined as another major oxidative stress response mechanism involving an interplay between the UPS and autophagy.

NRF2 is a transcription factor, and when activated, is upregulated antioxidant and metabolic enzymes, including TXNRD1 Suvorova et al.

KEAP1 is an adaptor protein of the E3 ligase Cullin-3 and plays a role in substrate recognition. Under normal conditions, transcription factor NRF2 was found in association with KEAP1-Cullin-3 E3 ligase complex, that catalyzed its ubiquitylation, rendering it a substrate for proteasomal elimination by selective autophagy Ishimura et al.

Competition resulted in the migration of free NRF2 to the nucleus and transactivation of stress-related cytoprotective genes Kobayashi et al.

Additionally, the NRF2—KEAP1 pathway provides a positive feedback loop for autophagy. P62 was characterized as a direct transcriptional target of activated NRF2 Jain et al. Moreover, KEAP1 regulation by p62 was modulated by the E3 ligase TRIM NRF2 activation was negatively affected by TRIMmediated Klinked ubiquitylation of p62 Pan et al.

Crosstalk between autophagy and the UPS may change character under disease conditions, contribute to the pathogenesis of diseases and even affect their outcome. Degenerative diseases and cancer are examples of diseases that illustrate the interplay between the UPS and autophagy in the clearance of misfolded abnormal proteins Juenemann et al.

For example, Huntington Disease is caused by poly-glutamine extensions in a protein called Huntingtin Htt , leading to abnormal organization and eventual aggregation of the protein. Htt protein was shown to be ubiquitylated via K or Klinked ubiquitin chains Bhat et al. Mutant Htt clearance depended on both the UPS and autophagy in different experimental settings.

Mutant Htt aggregates were largely cleared by Kdependent autophagy mechanisms Renna et al. On the other hand, overexpression of Kspecific E3 ligase Ube3a, resulted in a UPS-dependent degradation of mutant proteins. Yet, cellular levels of E3 ligase was shown to decline in an age-dependent manner.

Therefore, in elderly people, accumulation of Klinked polyubiquitylated proteins might tip the balance toward clearance of protein aggregates by autophagy. A similar UPS switch was also observed in a CHIP-dependent manner Jana et al.

Yet, autophagy was still functional under these conditions, and could significantly eliminate these aggregates Bayraktar et al. Therefore, preferential elimination of mutant proteins by autophagy might tip the balance in favor of wild-type proteins and restore disease-related loss of cellular functions including UPS-related mechanisms.

The role of the crosstalk between the two systems is also prominent in the cancer context. For example, the Pregulated and cancer-related protein EI24, was introduced as a critical link between the UPS and autophagy Devkota et al. EI24 controlled the stability of E3 ligases TRIM41, TRIM2, and TRIM28 by the regulation of their autophagic degradation Devkota et al.

Cellular levels of other E3 ligases, namely MDM2 and TRAF2, were also regulated by EIcontrolled degradation, modulating p53 and mTOR pathways, respectively, and influencing cancer formation and progression Devkota et al.

Changes include, modulation of levels of E3 ligases such as MDM2 Haupt et al. Under these circumstances, dynamic and complex changes in the regulation of the degradative pathways should have dramatic effects that contribute to cancer-related alterations in the proteomic landscape of cells.

Autophagy-UPS crosstalk emerges as a critical factor that determines the success of disease treatment, chemotherapy is one striking example. For instance, proteasome inhibition by the chemotherapy agent bortezomib resulted in the accumulation of misfolded proteins and induced compensatory autophagy in cancer cells Obeng et al.

Under these circumstances, autophagic activity protected cancer cells from bortezomib-induced cell death, and inhibition of autophagy improved the outcome of chemotherapy. These dual autophagy-UPS targeting approaches also gave promising results in clinical trials Vogl et al.

Several companies are now developing drugs that modulate the UPS or autophagy [for example, Huang and Dixit, ]. Concepts and data that were discussed above and elsewhere indicate that, depending on the disease type and treatment strategy, the crosstalk between the UPS and autophagy should definitely be taken into account in these efforts.

Autophagy and the ubiquitin proteasome systems are major degradation systems in mammalian cells that allow recycling of cellular contents ranging from soluble proteins to intracellular organelles. Although their mode of action and their requirements for substrate recognition are different, there are several overlaps and interconnections between the UPS and autophagy pathways.

A prominent component of the crosstalk is the ubiquitin protein itself and ubiquitylation. Indeed, ubiquitin is a common signal for both the UPS and autophagy.

It was proposed that, ubiquitin chain type could determine the pathway of choice for protein degradation. Klinked ubiquitylation was introduced to be a signal for the UPS, whereas Klinked ubiquitylation directed proteins for autophagosomal degradation Herhaus and Dikic, Yet, a number of independent studies provided evidence that both ubiquitylation types could lead to autophagic degradation of substrates Wandel et al.

Moreover, recent studies underline the importance of ubiquitin phosphorylation as an event that increased the affinity of autophagy receptors for their targets during selective autophagy Kane et al.

Additionally, non-ubiquitin modifications e. were shown to affect protein degradation as well Hwang and Lee, Therefore, a barcode of ubiquitin and other modifications seem to prime proteins for one or the other degradation pathway and determine their fate.

As another level of regulation, deconjugating enzymes such as DUBs may counteract or redirect proteins for different degradation systems. E3 ligases emerged as important components of the UPS-autophagy switches.

For example, Cullin-3 Pintard et al. On the other hand, the same E3 ligase that might be able to generate different ubiquitin linkages under different conditions and on different substrates Chan et al.

A prominent example is the Parkin protein. During mitophagy although some of the proteins that are ubiquitylated by Parkin are degraded, other ubiquitylated proteins contribute to mitochondrial clustering and recognition by autophagy receptors. To date, factors or modifications that determine the substrate selectivity of Parkin are unknown.

Signaling switches involved in the regulated activation of one or the other system was shown to modify cellular responses to stress.

For example, NRF2 degradation by the UPS was controlled through pmediated KEAP1 elimination by autophagy Jain et al. Prevention of HIF1α degradation by the UPS, resulted in the expression of stress response genes, including autophagy genes, led to autophagy activation.

In another example, the UPS activity was required for NF-κB activation and NF-κB-mediated autophagy gene upregulation. Yet, autophagic degradation of NF-κB activators NIK and IKKs provided a negative feedback loop in the control in this context Qing et al.

Degradation of the components or regulators of one system by the other system was also reported. For example, proteasomes were defined as substrates of selective autophagy Marshall et al. Conversely, various autophagy proteins were ubiquitylated and degraded by the UPS in a regulated manner.

Therefore, checks and balances between the two systems exist, and these control mechanisms possibly allow remodeling of the cellular proteome under different conditions. Compensation mechanisms are also operational between the two systems.

Inhibition of the UPS generally upregulated autophagy, whereas failures in the autophagy system were associated with increased UPS activity, although inefficient compensation and failure in both systems were also observed under certain conditions Korolchuk et al.

Moreover, alternative protein degradation pathways, such as CMA and microautophagy might come into play under these conditions as well. Nevertheless, depending on the character of the target to be degraded, compensation mechanisms were less or more effective.

For example, large aggregates and whole organelles should be cleared by autophagy, but defective ribosomal products that could not be accumulated in stress granules were shown to be directed for proteasomal degradation.

Therefore for cellular homeostasis and for proper functioning of cells, ideally both systems should be fully operational. Data obtained so far demonstrate that crosstalk and communication between autophagy and the UPS generally rely on non-specialized and even indirect links.

Yet, there might exist so far unknown specialized proteins providing coordination and co-regulation of the two systems. Furthermore, regulation through direct protein-protein interactions between known system components is another possibility.

Therefore, dedicated communication proteins or pathways between the degradation mechanisms may be present, allowing better and faster coordination in case of need. Further studies are required to unveil the nature of these putative proteins, interactions and pathways.

An emerging theme in the regulation and coordination of autophagy and the UPS involves non-coding RNAs and their intricate networks. A growing list of microRNAs as well as long non-coding RNAs were implicated in the control of autophagy Tekirdag et al. MicroRNAs have the advantage of affecting the level of multiple proteins at once, and they are able to rapidly reshape cellular signaling mechanisms and pathways.

Therefore, non-coding RNA networks possibly contribute to the co-regulation of these degradative systems. Intriguingly, deregulation of non-coding RNA levels contribute to the progression of diseases such as cancer.

Future studies on non-coding RNAs will reveal their relevance in the autophagy-UPS crosstalk under physiological and pathological conditions.

Overall, coordination, interconnection and crosstalk mechanisms between the UPS and autophagy exist at various levels. In addition to ubiquitin and ubiquitylation, several proteins and signaling pathways were implicated in the communication and mutual regulation of the two systems.

Considering the importance of protein catabolism for cellular and organismal homeostasis and health, a better understanding of individual systems as well as the interconnections and crosstalks between them will be most rewarding from both a basic science perspective and with regards to clinical management of diseases involving protein quality control problems.

NK and DG wrote the manuscript and did critical reading. NK prepared the illustrations in the manuscript. This work was supported by the Scientific and Technological Research Council of Turkey TÜBİTAK Grant Project Number T and Sabanci University.

NK was supported by TUBITAK-BIDEB A Ph. Scholarship during Ph. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Cell Sci. Devkota, S. Functional characterization of EIinduced autophagy in the degradation of RING-domain E3 ligases. AUTAC molecules consist of a POI-targeting warhead, a linker, and a cGMP-based degradation tag. The degradation tag recruits autophagosomes to degrade cytoplasmic proteins and cellular organelles.

In addition to cytoplasmic proteins, cellular organelles such as mitochondria could be degraded via AUTAC.

Takahashi et al developed a molecule known as AUTAC4, which promotes mitophagy of fragmented mitochondria. The treatment of AUTAC4 was shown to restore mitochondrial membrane potential and ATP production.

Similar to the autophagy-based AUTAC, autophagosome tethering compound ATTEC functions by tethering the POI to the autophagosome 96 , 97 Figs.

Whereas AUTAC recruits autophagosomes for degradation, ATTEC binds to LC3, one of the key proteins of autophagosome. Mutant huntingtin protein has at least 36 glutamines.

The longer the polyQ stretch, the earlier symptoms typically appear. Furthermore, it will be interesting to determine whether these ATTEC molecules can be used for other polyQ diseases, such as dentatorubral pallidoluysian atrophy and Machado-Joseph disease.

Schematic representation of ATTEC and AUTOTAC. An ATTEC molecule simultaneously binds LC3 and a POI, while an AUTOTAC molecule binds p62 and a POI. The binding induces the formation of autophagosomes, and subsequent fusion between autophagosomes and lysosomes lead to the POI degradation.

Recently, Lu and colleagues further extend the application of ATTEC by developing small molecules targeting Lipid droplets LD-ATTEC , the fat-storage organelles in cells.

Furthermore, they can rescue LD-related phenotypes in two independent mouse models. Such a conformation change exposes the LIR motif of p62, and prmotes its interaction with LC3 on the autophagic membrane. Ji et al. designed the AUTOphagy-TArgeting Chimera AUTOTAC platform that bypasses the requirement of ubiquitin 80 Fig.

AUTOTAC molecules consist of a module that interacts with the ZZ domain of p62, and a POI-targeting module. AUTOTAC promotes the oligomerization and activation of p62, leading to the degradation of the POI by the autophagy—lysosome pathway Fig.

AUTOTAC can mediate the targeted degradation of not only monomeric proteins, but also aggregation prone proteins. Using murine models expressing human pathological tau mutants, Ji et al demonstrated the AUTOTAC could effectively remove misfolded tau.

In addition to Tau, AUTOTACs could also efficiently remove multiple oncoproteins, such as degrading androgen receptor AR. In chaperone-mediated autophagy, heat shock protein 70 HSC70 recognizes soluble protein substrates with KFERQ sequence. CMA-based degraders include three functional domains: a cell membrane penetration sequence, a POI-binding sequence, and a CMA-targeting motif Fig.

Upon the addition to the cells, a CMA-based degrader first enters the cell, then binds to the target protein via the POI-binding sequence, and finally transports to the lysosomes for degradation. Schematic representation of CMA-based degrader. CMA-based degrader consists of three modules: a CMA-targeting module, a cell-penetrating peptide, and a POI-targeting module.

After the CMA-based degrader entering the cell, it binds the POI and induces chaperone-mediated autophagy. To become an effective therapeutic strategy, CMA-based degraders need to overcome at least two major hurdles. First, the stability of the degrader.

Second, the delivery efficiency. Overall, whereas the CMA-based degraders represent a new approach in TPD, they face great challenges that are not seen by other TPD technologies, such as PROTAC and LYTAC. The last few years have seen explosive growth in the field of TPD.

Multiple TPD molecules mostly based-on the PROTAC technology have shown potential therapeutic effects in cancer clinical trials and preclinical studies.

The estrogen receptor ER is a master regulator of gene expression, and is critical for the pathogenesis of breast cancer.

In clinical experiments, ARV shows good oral bioavailability and favorable tolerability. In addition to ARV, ARV is another PROTAC small molecule entering phase II clinical trial.

ARV is developed as a potential treatment for prostate cancer, the second most common malignancy in men after lung cancer. Signal transducer and activator of transcription 3 STAT3 is constitutively activated in a variety of human cancers. Recently, a potent and specific PROTAC degrader of STAT3, SD, was developed.

Interestingly, SD is highly selective for STAT3 among all the STAT proteins. Furthermore, SD also achieves a robust and long-lasting STAT3 degradation in multiple xenograft mouse models.

BCL-XL, a member of the BCL-2 family, protects cancer cells from programmed cell death. Recently, a potent BCL-XL PROTAC molecule, DT, was developed by conjugation ABT with a VHL ligand. It also displays reduced side effects relative to ABT, likely due to the low expression of VHL in platelets.

Interestingly, DT, although binding to BCL-XL and BCL-2 with similar affinity, does not induce the degradation of BCL Neurodegenerative diseases NDs are a group of disorders characterized by progressive motor or cognitive impairment.

As a result, novel drug discovery modes, such as TPD, are urgently needed in order to develop therapeutic approaches for NDs. In , Chen and Li groups reported a PROTAC molecule targeting tau protein, the first attempt to apply the PROTAC technology for the treatment of NDs.

Another polypeptide PROTAC for AD was developed by Jiang et al. In addition to cancer and neurodegenerative diseases, the reach of TPD has extended to inflammatory diseases and immuno-oncology.

IRAK-4 interleukin-1 receptor-associated kinase 4 is a member of the IRAK kinase family and involved in Toll-like receptor TLR and IL-1R signaling pathways.

Indeed, multiple IRAKtargeting PROTAC molecules have been developed, with one entering phase I clinical trials to treat autoimmune diseases. BTK is an established target in both inflammation and cancer.

These challenges could be uniquely addressed by BTK degraders as these molecules may degrade both wide-type and mutant BTK proteins. Viral infection poses a great challenge in global health. SARS-CoV-2 is one of the worst examples, which have infected over million individual and killed 5.

de Wispelaere et al. Currently, there are great interests in the development of PROTACs that target SARS-CoV-2 across academia and industry. The past two decades have seen the birth and boom of the TPD technologies. PROTAC and molecular glue are the most advanced TPD technology. Both are based on the ubiquitin-proteasome system and useful for the degradation of intracellular proteins.

In the past 5 years, technologies harnessing the second degradation pathway in cells have emerged and quickly developed. These technologies can be further divided into two groups based on their degradation mechanisms.

LYTAC, Bispecific Aptamer Chimeras, AbTAC, and GlueTAC, degrade extracellular and membrane proteins by harnessing the endosome-lysosome pathway. In addition, technologies targeting the autophagy-lysosome pathway, such as AUTAC, ATTEC, AUTOTAC, and CMA chimeras, can degrade misfolded proteins, protein aggregation, or damaged organelles.

Multiple PROTAC molecules, including cancer drug candidates ARV and ARV, have shown great promising in clinical trials. Nevertheless, the PROTAC technology, as a whole, still faces many challenges. First of all, pharmaceutical properties.

PROTAC molecules often face the challenges of cell permeability and oral bioavailability due to their large size. Molecular glues are smaller and have some advantages over PROTAC molecules; however, they are more difficult to rationally design.

Second, the repertory of E3 ubiquitin ligase. Human genome encodes more than E3 ubiquitin ligases, and only a few of them VHL, CRBN, IAPs, and MDM2 have been utilized to degrade target proteins.

Third, toxicity. PROTAC could result in more toxicity than small molecular inhibitors because they degrade entire targeted proteins, rather than solely inhibit them. Relative to PROTAC and molecular glue, the development of lysosome-based TPD technologies is still in the infancy stage.

We still have much to learn about the specific mechanism of each technology. As an important intracellular organelle, lysosomes regulate many important cellular and physiological functions in addition to protein degradation, such as the metabolism and homeostasis.

Expanding the repertory of lysosome-targeting receptors, which currently include CI-MPR and ASGPR only, is much needed for LYTAC and similar technologies. These efforts will help to develop the autophagy-based technologies as a general method for protein degradation, analogous to PROTAC.

Current CMA-based degraders are mostly limited by cell membrane permeability and stability, and their small-molecule forms may overcome these obstacles.

Lysosome-based technologies have greatly broadened the spectrum of targets by PROTAC and molecular glue, and a surge of interest in this field is definitely expected.

Despite these challenges, TPD technologies, undoubtedly, will not only provide powerful tools for biomedical research, but hold great promise for future drug development.

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