bims-proteo Biomed News
on Proteostasis
Issue of 2022‒05‒15
forty papers selected by
Eric Chevet

  1. FASEB J. 2022 May;36 Suppl 1
      Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as endoplasmic reticulum associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition controls ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long chain ceramides leads to a dramatic increase in the level of ubiquitinated substrates in the ER membrane, and concomitantly, reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized regulatory mechanism in which ER membrane lipid remodeling controls the ERAD system.
  2. FASEB J. 2022 May;36 Suppl 1
      Protein quality control (PQC) maintains proteostasis in cells, whereas PQC dysregulation is found in diseases such as cancers, cardiovascular diseases, and neurodegenerative disorders. Over the past few decades, remarkable progress has been made in understanding compartmentalized PQC pathways that operate inside the nucleus and the endoplasmic reticulum (ER). The small ubiquitin-related modifiers (SUMOs), for example, were recently found to participate in a nuclear PQC pathway that involves selective sumoylation of misfolded proteins by the SUMO E3 ligase PML, and subsequent recognition and ubiquitylation by the SUMO-targeted ubiquitin E3 ligase RNF4 (Guo et al., Mol Cell, 2014). Roles of SUMOs in cytoplasmic PQC, however, have not previously been reported. Using yeast mutants and human knockout cell lines, our lab has recently identified a conserved SUMO-dependent pathway for degradation of a cytosolic, misfolded protein model substrate. The substrate consists of GFP fused to an N-terminal nuclear export signal (NES) and a C-terminal hydrophobic degron known as CL1 (NES-GFP-CL1). Using protein stability assays, we observed a decrease in the turnover rate of NES-GFP-CL1 in a yeast SUMO mutant strain and a human U2OS SUMO1 knockout cell line. Further exploration using nuclear and ER-localized substrates revealed that this pathway is specific for soluble, cytosolic proteins. In addition, analysis of a U2OS SUMO2 knockout cell line revealed that the observed PQC pathway is specific for the SUMO1 paralogue. This suggests a distinct mechanism from the previously characterized nuclear PML-SUMO-RNF4 pathway, which targets misfolded proteins modified by SUMO2/3 polymeric chains. Using cellular fractionation studies, we observed an increase in levels of insoluble NES-GFP-CL1 specifically in SUMO1 knockout cells compared to wild type cells. We therefore propose a model where SUMO1 modification of misfolded cytosolic proteins promotes their turnover by enhancing solubility and preventing aggregation. Results from our studies reveal a novel cytosolic PQC regulatory network and provide insights into roles for sumoylation in PQC-associated diseases.
  3. FASEB J. 2022 May;36 Suppl 1
      Producing diverse membrane proteins requires multi-step biosynthetic pathways with tightly integrated quality control mechanisms. Two biosynthetic challenges common to all membrane proteins are the need to localize to the appropriate cellular membrane and the need to insert each transmembrane segment (TM) in the correct topology. Using approaches that integrate cell biology, structural biology, and biochemistry, we examine mechanisms that ensure the fidelity of single-spanning membrane protein localization and topogenesis at the endoplasmic reticulum (ER). We recently discovered that the ER-resident P5A-ATPase ATP13A1 (yeast Spf1) dislocates mislocalized mitochondrial tail-anchored (TA) proteins. We leverage ATP13A1 as an experimental handle to investigate how mitochondrial membrane proteins misinsert into the ER. We also identify a role for ATP13A1 in the topogenesis of type II membrane proteins that should insert into the ER membrane with their N-terminus in the cytosol. Without corrective protein quality control by ATP13A1, mislocalized TA proteins and misinserted type II proteins engage distinct ER-associated degradation (ERAD) mechanisms. Our findings reveal- how cells integrate counteracting ER activities with different specificities to fine tune membrane protein biogenesis.
  4. FASEB J. 2022 May;36 Suppl 1
      To maintain protein homeostasis (i.e., "proteostasis") and withstand the toxic effects brought about by the presence of misfolded proteins, eukaryotes have evolved a hierarchy of quality control checkpoints along the secretory pathway. The most prominent quality control step in this pathway, which acts during or soon after proteins are synthesized, is endoplasmic reticulum associated degradation (ERAD). The importance of this pathway is underscored by the fact that ~80 different protein substrates of the ERAD pathway have been linked to human disease. Although most misfolded proteins in the secretory pathway are eliminated by ERAD, others can exit the ER in COPII vesicles and are instead turned over by lysosomal proteases. This post-ER quality control event requires the ESCRT machinery. A different class of secreted proteins, particularly those that are aggregation-prone, can alternatively be degraded by ER-phagy. To date, it remains elusive how these diverse misfolded proteins--which can trigger various stress responses--are selected for different fates. However, by constructing a collection of model substrates and examining wild-type and disease-associated mutant forms of various proteins, we are beginning to define the requirements for the targeted selection of misfolded proteins in the secretory pathway for one fate versus another. This pursuit represents a vital step toward the development of pharmaceuticals that might one day repair folding-defective proteins. Indeed, a growing number of clinical and pre-clinical drugs that repair ERAD and other quality control substrates have shown efficacy in various disease models. In this presentation, each of these topics will be discussed and future research directions defined.
  5. Bio Protoc. 2022 Apr 05. 12(7): e4368
      The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major protein degradation routes for maintaining cellular protein homeostasis, and ubiquitin E3 ligases, components of ubiquitin ligase complexes, play an important role in the selective degradation of target proteins via substrate-specific interactions. Thus, understanding the role of E3 ligases and their substrate regulation uncovers their specific cellular and physiological functions. Here, we provide protocols for auto- and substrate-ubiquitination analyses that utilize the combination of in vitro purified E3 ubiquitin ligase proteins and immunoprecipitation.
    Keywords:  ACC synthase; SINAT2; Self-ubiquitination; Substrate-ubiquitination; Ubiquitination
  6. FASEB J. 2022 May;36 Suppl 1
      Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane where they are correctly folded, assembled, and then delivered to their final cellular destinations. In order to prevent the accumulation of misfolded membrane proteins, ER associated degradation (ERAD) moves these clients from the ER membrane to the cytosol; a process known as retrotranslocation. Our recent work in S. cerevisiae has revealed a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. We have sought to understand the mechanism associated with Dfm1's actions and found that Dfm1's conserved rhomboid residues are critical for membrane protein retrotranslocation. Specifically, we identified several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 has retained the lipid thinning functions of its rhomboid protease predecessors to facilitate in the removal of ER membrane substrates. We find this substrate engagement and lipid thinning feature is conserved in its human homolog, Derlin-1. Utilizing interaction studies and molecular dynamic simulations, this work reveals that rhomboid pseudoprotease derlins employ novel mechanisms of substrate engagement and lipid thinning for catalyzing extraction of multi-spanning membrane substrates.
  7. FASEB J. 2022 May;36 Suppl 1
      Neurodegenerative diseases afflict over 5 million Americans and are caused primarily by proteins and protein aggregates that disrupt proteostasis; the process which maintains protein function and quality control in the cell. Healthy cells can process small aggregates through the action of molecular chaperone assemblies and protein degradation pathways. However, highly stable aggregates irreversibly disrupt proteostasis and trigger disease onset. In contrast to human cells, the chaperone Hsp104 can resolve highly stable aggregates in yeast. Problematically, humans lack Hsp104. Therefore, we hypothesize that metazoan cells have developed alternative machinery to resolve stable protein aggregates. To address this hypothesis, we developed multiple endoplasmic reticulum (ER) localized substrates that have aggregation-prone cytosolic motifs. Substrates were comprised of either yeast or mammalian protein-derived membrane anchors and aggregation-prone and amyloid-like motifs. Because of their structure, we hypothesized that these substrates were dependent on ER associated degradation (ERAD) and became insoluble under stress conditions. We evaluated these substrates in HEK293H cells with cycloheximide chase and detergent fractionation assays and discovered each substrate largely depended on the proteasome for degradation while only some were insoluble at elevated temperatures. We then used biotin proximity labeling to identify potential protein chaperones associated with our substrates.
  8. Life Sci. 2022 May 06. pii: S0024-3205(22)00320-4. [Epub ahead of print] 120620
      In tumor cells, the endoplasmic reticulum (ER) plays an essential role in maintaining cellular proteostasis by stimulating unfolded protein response (UPR) underlying stress conditions. ER-associated degradation (ERAD) is a critical pathway of the UPR to protect cells from ER stress-induced apoptosis and the elimination of unfolded or misfolded proteins by the ubiquitin-proteasome system (UPS). 3-Hydroxy-3-methylglutaryl reductase degradation (HRD1) as an E3 ubiquitin ligase plays an essential role in the ubiquitination and dislocation of misfolded protein in ERAD. In addition, HRD1 can target other normal folded proteins. In various types of cancer, the expression of HRD1 is dysregulated, and it targets different molecules to develop cancer hallmarks or suppress the progression of the disease. Recent investigations have defined the role of HRD1 in drug resistance in types of cancer. This review focuses on the molecular mechanisms of HRD1 and its roles in cancer pathogenesis and discusses the worthiness of targeting HRD1 as a novel therapeutic strategy in cancer.
    Keywords:  Cancer; HRD1; Therapy resistance; Ubiquitination
  9. FASEB J. 2022 May;36 Suppl 1
      Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress-induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi-pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome-level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock-out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3-lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock-in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.
  10. FASEB J. 2022 May;36 Suppl 1
      Stress granules are dynamic biomolecular condensates of proteins and non-translating RNAs that form when translation is inhibited during stress. Stress conditions associated with disease including ER dysfunction, toxic metalloids, and inflammatory factors induce stress granule formation. Stress granules disassemble when translation resumes during the recovery from stress. Aberrant, cytotoxic stress granules are implicated in degenerative diseases of the nervous, muscular and skeletal systems. Yet, the mechanisms underlying stress granule dynamicity are not well understood. A growing body of research suggests that the ubiquitin-proteasome system regulates the formation and disassembly of stress granules. Valosin-containing protein (VCP) is a homohexameric AAA+ ATPase that functions as a ubiquitin-binding protein segregase in protein quality control and general protein degradation pathways in the ubiquitin-proteasome system. Prior research demonstrated that VCP is an important mediator of stress granule disassembly. Our recent work suggests an additional role for VCP in stress granule assembly. By imaging single mRNA molecules in living and fixed human cells, we determined that VCP and other members of the ribosome-associated quality control pathway (i.e. listerin, nuclear export mediator factor, and the proteasome) are critical for the release of specific mRNAs from translation complexes and localization to stress granules. Overexpression of VCP alleles associated with amyotrophic lateral sclerosis and frontotemporal dementia caused increased mRNA localization to stress granules. Thus, understanding the molecular mechanisms by which VCP regulates stress granules will be important for discovering its role in disease. This research will contribute to our knowledge of how defects in stress granules and the ubiquitin-proteasome system drive human degenerative diseases and suggest potential diagnostic and therapeutic strategies.
  11. FASEB J. 2022 May;36 Suppl 1
      The integrated stress response (ISR) and the unfolded protein response (UPR) are conserved signaling networks governed by stress sensor kinases. Four ISR sensor kinases, GCN2, HRI, PERK, and PKR, phosphorylate eIF2α in response to multiple stresses, leading to a global protein synthesis shutdown coupled to the translation of select mRNAs, which results in an adaptive remodeling of the proteome. Besides PERK, which the ISR and the UPR share, the UPR relies on two additional ER stress sensors, the kinase/RNase IRE1 and the membrane-tethered transcription factor ATF6, to induce adaptive signaling programs that reinstate ER homeostasis. However, if the stress is unrelenting, both the ISR and the UPR can switch to drive cell death. The mechanistic commonalities and crosstalk between the ISR and UPR remain an active area of investigation. In this talk, I will share new results that support common signaling principles of the ISR and UPR sensor kinases and reveal a new layer of communication between the ISR and the UPR. Specifically, we found that dynamic clustering is a prominent feature of PKR activation reminiscent of the high-order assemblies of IRE1 and PERK observed during ER stress. Surprisingly, PKR clusters excluded eIF2α, and mutations in PKR that disrupt cluster assembly enhanced eIF2α phosphorylation, suggesting that PKR clusters act as enzyme sinks that control enzyme-substrate interactions by limiting PKR-eIF2α encounters. Moreover, stress-free activation of PKR induced a master cell death program dependent on ISR-driven expression of DR5 (death receptor 5), as occurs during unmitigable ER stress. Remarkably, stress-free activation of the ISR selectively activated IRE1 independent of sensing unfolded proteins in the ER lumen, and treatment with the small-molecule ISR inhibitor ISRIB reversed it. Our data provide new mechanistic insights into two fundamental aspects of stress sensor kinase signaling: (1) dynamic clustering of stress sensors may provide the means to fine-tune stress responses, and (2) the ISR selectively activates IRE1, thus coupling the ISR and the UPR outside their common node PERK.
  12. FASEB J. 2022 May;36 Suppl 1
      Maintaining cellular proteostasis is challenging due to the constantly changing and crowded cellular environment. Mutations, transcriptional and translational errors, and chemical and pathological stresses in a cell lead to protein misfolding and eventual formation of insoluble toxic aggregates. These aggregates typically result in loss of native protein function and are often associated with conformational diseases such as Alzheimer's, Parkinson's, Huntington's, and various types of cancer. The cellular protein quality control systems are essential for creating a stable cellular environment by resolving the misfolded proteins to uphold a stable proteome. The triage decision for a misfolded protein is regulated by Heat shock protein 70 (Hsp70), the central hub of protein quality control machinery, along with the E3 ubiquitin ligase C-terminus of Hsc70-interacting protein (CHIP). Hsp70 itself helps misfolded "client" proteins to refold and directs protein clients to downstream refolding pathways, whereas Hsp70 interaction with CHIP can lead to CHIP-mediated ubiquitination, ultimately directing the proteins to proteasomal degradation. Understanding the molecular mechanisms that enable the Hsp70/CHIP complex to triage client proteins would allow for the identification of new avenues of treatment for the above-mentioned diseases. With an objective to illustrate how the varying degrees of folding in a model client protein dictate the interaction between Hsp70 and the client, we have designed a spectrum of client proteins we named folding sensor TPRs (FSTPRs). We initially hypothesized that the more unfolded the client higher the affinity towards Hsp70. Circular dichroism (CD) spectroscopy was used to assess the folding states of each FSTPR variant, and biolayer interferometry (BLI) was utilized to quantify the affinities of each FSTPR towards the Hsp70 substrate-binding domain (SBD). During our BLI experiments, we observed a range of threshold unfolding percentages that correspond to the highest affinity. Above and below the threshold range, the affinity decreased. This behavior was recapitulated with a disulfide-locked FSTPR that is folded in the oxidized state and predominantly unfolded in the reduced state. Even though support from further data is necessary, these studies provide us preliminary insight into how Hsp70 interacts with a misfolded client and the role played by folding state in dictating Hsp70/client interactions.
  13. FASEB J. 2022 May;36 Suppl 1
      The unfolded protein response (UPR) is sensitive to both proteotoxic stress and membrane bilayer stress. These stresses are sensed by the ER transmembrane protein Ire1. When activated, Ire1 uses its endonuclease activity to splice HAC1mRNA, producing a mature transcription factor that binds to UPR elements (UPREs) in the promoters of target genes. Hac1 targets include not only genes involved in protein folding, secretion, and degradation, but also a subset of lipid metabolic genes. One aspect of lipid metabolism is the deacylation of phosphatidylcholine (PC) by phospholipases to produce glycerophosphocholine (GPC). In Saccharomyces cerevisiae, GPC can be reacylated in a novel two-step process catalyzed first by GPC acyltransferase Gpc1, followed by acylation of the lyso-PC molecule by Ale1. This metabolic cycle has been termed the PC deacylation/reacylation pathway (PC-DRP). In prior studies, loss of Gpc1 was shown to result in an increase in di-unsaturated PC species at the expense of mono-unsaturated PC species, indicating a role for PC-DRP in PC acyl chain remodeling. Here, we probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR-inducing compounds tunicamycin, DTT, and canavanine results in an increase in GPC1 message that is dependent upon the UPR transcriptional activator Hac1. The importance of this increased expression to cellular function is illustrated by the finding that cells lacking Gpc1 exhibit increased sensitivity to those compounds. In a converse set of experiments, we show that that loss of GPC1 results in upregulation of the UPR as measured by expression of the ER chaperone KAR2. Consistent with these findings, we show that Gpc1 primarily co-localizes with the endoplasmic reticulum.
  14. FASEB J. 2022 May;36 Suppl 1
      Protein modifications by ubiquitin and ubiquitin-like proteins are emerging as an important mechanism regulating heart function. UFM1 (ubiquitin-fold modifier 1) is a novel ubiquitin-like protein that modifies protein targets via UFM1-specific conjugation enzymes. Dysregulation of UFM1 modification, termed ufmylation, has been linked to multiple human diseases but its importance in the heart remains unclear. We have previously reported that ufmylation is upregulated in compensated, hypertrophic mouse hearts but decreased in failing human hearts. Inhibition of ufmylation by targeted ablation of the E3 UFM1 ligase 1 (UFL1) in mouse hearts resulted in dilated cardiomyopathy during ageing and increased the propensity to heart failure in response to hemodynamic stress. However, how ufmylation exerts its cardioprotective effect remains unclear. Here, we report that UFBP1 (UFM1 binding protein 1), an ER-resident ufmylation target, is an important downstream effector of ufmylation in the heart. While deletion of UFBP1 in mouse hearts has no impact on ufmylation, the knockout (KO) mice developed dilated cardiomyopathy at resting condition, as indicated by left ventricular wall thinning, chamber dilatation and significantly decreased cardiac contractility at 6 months of age. Moreover, loss of UFBP1 exacerbated pressure overload-induced cardiac remodeling and dysfunction, recapitulating multiple aspects of the phenotypes of UFL1-deficient hearts. Mechanistically, UFL1 controls the expression of UFBP1. Depletion of both UFL1 and UFBP1 in cultured cardiomyocytes aggravated ER stress-induced cell injury. Furthermore, excess ER stress is implicated in UFBP1KO hearts and is aggravated with the progression of cardiomyopathy. Interestingly, ER stress inducers upregulated the expression of ufmylation pathway components at both transcript and protein levels in cultured cardiomyocytes and mouse hearts, indicating an intimate cross-talk between ufmylation and ER stress. Collectively, our data identify a novel UFM1-UFL1-UFBP1 axis in constraining pathological cardiac remodeling and maintaining ER homeostasis.
  15. Autophagy. 2022 May 09.
      A recent screen of the Saccharomyces cerevisiae deletion library implicated End3 in autophagy of the endoplasmic reticulum (ER). Together with Pan1, End3 coordinates endocytic site initiation with the localized assembly of branching actin filaments that promotes invagination of endocytic pits. Oxysterol binding proteins function as an inter-organelle bridge by interacting with VAP proteins on the cortical ER and type I myosins on the endocytic pit. These proteins not only promote localized actin assembly at contact sites, they are required for ER autophagy as well. We propose that localized actin polymerization can push the edge of an ER sheet from the cell cortex towards the site of autophagosome assembly near the vacuole.
    Keywords:  Actin assembly; End3-Pan1; Myo3/Myo5; Osh2/Osh3; Scs2/Scs22; contact sites; endocytic pits; endoplasmic reticulum; reticulophagy
  16. FASEB J. 2022 May;36 Suppl 1
      GRP170 is an Hsp70-like, molecular chaperone localized to the endoplasmic reticulum (ER). Two separate functions have been described for GRP170. First, GRP170 acts as a nucleotide exchange factor (co-chaperone) for the ER lumenal, Hsp70, BiP. Second, GRP170 possess "holdase" activity, and independently binds to aggregation prone regions of proteins to maintain solubility. We previously demonstrated that GRP170 regulates the quality control of the epithelial sodium channel, ENaC. ENaC is responsible for sodium reabsorption in the distal nephron and regulates salt/water homeostasis, and therefore, blood pressure. To better understand how GRP170 impacts kidney function we generated an inducible, nephron specific, GRP170 KO mouse. Loss of GRP170 results in rapid weight/volume loss, electrolyte imbalance and significantly elevated aldosterone levels. The GRP170 KO animals also demonstrate many of the hallmarks of acute kidney injury (AKI) including elevated plasma BUN and creatinine levels. Loss of GRP170 also results in induction of the unfolded protein response (UPR) as shown by upregulation of UPR targets by qPCR (sXbp1, BiP, CHOP and ATF4) and western blotting. We hypothesize that sustained induction of the UPR leads to kidney injury associated with our model, alternatively, misregulation of ion channel trafficking and volume loss may contribute to the pathogenic phenotype. To begin to understand how loss of an ER localized chaperone results in profound kidney injury we treated our GRP170 KO mice with the UPR inhibitor, TUDCA, or a high-salt diet. Preliminary data suggest both UPR induction and electrolyte imbalance may contribute to kidney injury associated with loss of GRP170.
  17. FASEB J. 2022 May;36 Suppl 1
      The export of newly synthesized membrane proteins from the endoplasmic reticulum (ER) requires the action of coat protein complex II (COPII), which is comprised of five core subunits (Sar1, Sec23, Sec24, Sec13, and Sec31) that co-assemble to remodel membranes and generate transport carriers. Recent studies conducted in our lab demonstrate that COPII carrier biogenesis surprisingly continues in the absence of Sar1 GTPase activity, which was suggested previously to be indispensable for COPII-mediated membrane reorganization. Specifically, we show that inhibition of Sar1 stimulates ER stress and triggers the formation of unconventional transport carriers that are decorated with other COPII components and remain capable of directing secretory protein trafficking out of the ER. Our studies suggest new mechanisms by which COPII drives membrane remodeling independently of Sar1.
  18. FASEB J. 2022 May;36 Suppl 1
      The covalent attachment of ubiquitin to substrates controls virtually all aspects of the cell. With rare exceptions, ubiquitin was understood to exert its effects by becoming attached to the amino group of lysine residues within protein substrates. Recent discoveries from our lab and others have revealed that dedicated writers (E3 ligases) and erasers (DUBs) of non-lysine ubiquitination are intrinsic to eukaryotes. This highlights that attachment to sites beyond lysine are physiologically and perhaps pathologically important. These E3s tend to be highly divergent from their established counterparts and "activity-based" chemical biology approaches have been instrumental in identifying them. I will present work from my lab on technologies we have used to uncover unusual E3 ligases and the striking nature of their mechanism and substrate specificity. I will also discuss our characterisation of a small DUB family that is highly selective at removing ubiquitin from hydroxy amino acids. These new developments indicate that non-lysine ubiquitination is an integral component of the ubiquitin system that is subject to sophisticated regulation.
  19. J Cell Sci. 2022 May 01. pii: jcs259596. [Epub ahead of print]135(9):
      The heterotrimeric BAG6 complex coordinates the direct handover of newly synthesised tail-anchored (TA) membrane proteins from an SGTA-bound preloading complex to the endoplasmic reticulum (ER) delivery component TRC40. In contrast, defective precursors, including aberrant TA proteins, form a stable complex with this cytosolic protein quality control factor, enabling such clients to be either productively re-routed or selectively degraded. We identify the mitochondrial antiviral-signalling protein (MAVS) as an endogenous TA client of both SGTA and the BAG6 complex. Our data suggest that the BAG6 complex binds to a cytosolic pool of MAVS before its misinsertion into the ER membrane, from where it can subsequently be removed via ATP13A1-mediated dislocation. This BAG6-associated fraction of MAVS is dynamic and responds to the activation of an innate immune response, suggesting that BAG6 may modulate the pool of MAVS that is available for coordinating the cellular response to viral infection.
    Keywords:  BioID2; ER membrane complex; Protein targeting; SGTA; Tail-anchored proteins
  20. FASEB J. 2022 May;36 Suppl 1
      Lipid droplets (LDs) are neutral lipid containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain of these proteins is similar to a reticulon homology domain. Like reticulons, MCTPs tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the transmembrane domains (TMD) of MCTP promote LD biogenesis by increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form specialized subdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae are associated with other organelles as well, suggesting that these proteins may play a more general role in linking tubular ER to organelle contact sites.
  21. Curr Opin Cell Biol. 2022 May 06. pii: S0955-0674(22)00030-8. [Epub ahead of print]76 102084
      Autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is responsible for the degradation of ER portions by lysosomes. ER-phagy is induced in both physiological and stress conditions to maintain ER homeostasis and protein quality control. ER-phagy receptors and their interactors are key regulators of this process. Transcriptional and post-translational regulation of ER-phagy receptors have emerged as critical mechanisms for the modulation of ER-phagy, providing the first hints to understand how this process responds to the cellular needs. Here, we concisely review the main mechanisms regulating ER-phagy receptors and discuss their potential implications in diseases.
  22. FASEB J. 2022 May;36 Suppl 1
      Excessive inflammation underlies many human diseases, such as neurodegeneration, autoimmune disorders, cancer, and COVID-19. Thus, cellular mechanisms that control inflammation are of high therapeutic interest. Autophagy has been implicated in the suppression of inflammation, yet mechanistic links between autophagy and inflammation are not completely understood. Previous work demonstrated that loss of ATG9A, but not ATG5 or ATG7, increased inflammatory signaling through the STING-IRF3 cascade, suggesting that perhaps an autophagy-independent function of ATG9A regulates inflammation.1 Here, we show that loss of the essential basal autophagy regulators ATG9A and ATG101 sensitizes cells to dsDNA-induced IRF3 activation. Importantly, this effect was not observed with the loss of ATG5 or ATG7, suggesting that the canonical LC3-lipidation autophagy machinery is not required for suppression of IRF3 and inflammatory signaling.2 In an effort to understand the role of ATG9A and ATG101 in suppressing inflammatory signaling, we found that loss of ATG9A and ATG101, but not ATG5 or ATG7, caused an accumulation of ubiquitin-rich condensates. We also found that the accumulation of ubiquitin-rich condensates coincided with an overactivation of the ubiquitin-sensing, IRF3-targeted kinase TBK1. Importantly, we found that inhibiting the accumulation of ubiquitin-rich condensates via knock-out of p62/SQSTM1 abrogated the increased inflammatory signaling caused by loss of ATG9A. Together, our data suggest a model in which the loss of basal autophagy machinery causes an accumulation of ubiquitin-rich condensates, which, in turn, act as a platform for the aberrant activation of TBK1-mediated inflammatory signaling. We propose that these data have important implications for diseases of protein aggregation wherein similar ubiquitin-rich condensates form and aberrant inflammatory signaling plays a pathological role. REFERENCES: (1) Saitoh, T.; Fujita, N.; Hayashi, T.; Takahara, K.; Satoh, T.; Lee, H.; Matsunaga, K.; Kageyama, S.; Omori, H.; Noda, T.; Yamamoto, N.; Kawai, T.; Ishii, K.; Takeuchi, O.; Yoshimori, T.; Akira, S. Atg9a Controls DsDNA-Driven Dynamic Translocation of STING and the Innate Immune Response. PNAS 2009, 106 (49). (2) Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L. BioID Reveals an ATG9A Interaction with ATG13-ATG101 in the Degradation of P62/SQSTM1-ubiquitin Clusters. EMBO reports 2021, 22 (10).
  23. FASEB J. 2022 May;36 Suppl 1
      During exposure to environmental stresses, eukaryotic cells must reprogram gene expression at the transcriptional and translational levels in order to thrive under these new conditions. Dysregulation of gene expression under stress can lead to molecular damage, cellular death, and the progression of diseases. Therefore, regulation of gene expression is dynamic, requires multiple layers of control, and is critical for cellular adaptation and survival. However, many of these control mechanisms, particularly at the translational level, remain elusive. In response to oxidative stress, we observed in budding yeast a massive accumulation of K63 ubiquitin conjugates that supports cellular resistance to stress. By developing a sequential enrichment methodology, our proteomics analysis revealed that ribosomal proteins are the main targets of K63 ubiquitination under stress. Moreover, we determine that accumulation of K63 ubiquitinated ribosomes relies on the activity of two redox-sensitive ubiquitin enzymes: the E2 conjugase Rad6 and the deubiquitinating enzyme Ubp2. Activity and cryo-EM structural analysis revealed that K63 ubiquitin modifies fully assembled monosomes and polysomes and is required for pausing translation at the elongation step. We named this pathway Redox control of Translation by Ubiquitin (RTU), a novel mechanism that supports cellular resistance to stress by aiding the reprogramming of gene expression at the translational level.
  24. FASEB J. 2022 May;36 Suppl 1
      Heat shock protein (Hsp) 70 is the main mediator of protein quality control in the cytosol of eukaryotic cells. One of the major functions of Hsp70 is mediate the ubiquitination of client proteins which subsequently leads to proteasomal degradation. This process occurs via a multiprotein complex composed of Hsp70, carboxy terminus of Hsc70 interacting protein (CHIP), ubiquitin-conjugating enzyme (E2), and ubiquitin (Ub). This complex is thought to be flexible, however, knowledge of the degree of dynamics are still lacking and detailed experimental evidence is needed. Here we present the models derived from Electron Paramagnetic Resonance (EPR) data and small-angle X-ray scattering (SAXS) ensembles for the complexes of Hsp70/CHIP/E2-Ub with ATP or ADP. The model of ATP bound Hsp70/CHIP/E2-Ub complex has an average distance of ~120 Å between the substrate-binding domain (SBD) and the E2-ubiquitin active site. The same distance of the multiprotein complex is found to be decreased to ~60 Å when Hsp70 is bound to ADP. Since the ADP bound conformation is considered as the active conformation that tightly binds client proteins, the alteration of the average distance between the Hsp70 SBD and the E2-Ub active site implies that the Lys residue of the substrate might need to be positioned within appropriate distance to span the gap in order to efficiently promote ubiquitination. Here, we used molecular dynamic simulations, double electron-electron resonance (DEER) EPR spectroscopy and in-vitroubiquitination assays with designed model substrate proteins that place lysine residues at variable positions. We found the dynamics of the Hsp70/CHIP/E2-Ubiquitin complex is playing a major role in the ubiquitination of various client proteins and these dynamics are necessary to overcome the distance barrier between E2-Ub active site and the Hsp70 SBD. Our data also suggests that the Hsp70/CHIP complex is capable of ubiquitinating a client protein substrate when lysine residues are placed at several disparate positions. In sum, our data reveals the functional dynamics of the Hsp70/CHIP/E2-Ub complex which enable ubiquitination of a client protein.
  25. FASEB J. 2022 May;36 Suppl 1
      The Ubiquitin-Proteasome System is responsible for the bulk of protein degradation in eukaryotic cells. Proteins are generally targeted to the 26S proteasome by the attachment of polyubiquitin chains. A number of proteins also contain ubiquitin-independent degrons (UbID) that allow for proteasomal targeting without the need for ubiquitination. UbID substrates are degraded less processively than ubiquitinated substrates, but the mechanism underlying this difference remains unclear. We therefore designed two model substrates containing both a ubiquitination site and a UbID for a more direct comparison. We found UbID degradation to be overall less robust with complete degradation only occurring with relatively unstable substrates (those that were at least transiently unfolded as determined by protease sensitivity). Surprisingly, UbID degradation was unaffected by the non-hydrolyzable ATP analog ATPγS, which halts ATP-dependent engagement and translocation of substrates. Furthermore, ubiquitin-dependent degradation proceeded in a strikingly similar fashion to UbID degradation in the presence of ATPγS, suggesting the 26S proteasome may have a "default" ATP- and ubiquitin-independent mechanism that is capable of unfolding and degrading less-stable proteins.
  26. FASEB J. 2022 May;36 Suppl 1
      The ERK1/2 signaling pathway is critical in organismal development and tissue morphogenesis. Deregulation of this pathway leads to congenital abnormalities with severe developmental dysmorphisms, i.e. RASopathies. The core ERK1/2 cascade relies on scaffold proteins such as Shoc2 to guide and fine-tune its signals. Mutations in shoc2 lead to the development of the pathology termed Noonan-like Syndrome with Loose Anagen Hair (NSLAH) RASopathy. However, the mechanisms underlying the functions of Shoc2 and its contributions to disease progression remain unclear. We found that Shoc2 assembles an elegant multi-component complex that incorporates several proteins of the ubiquitin system. To fine-tune the amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97, and the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism. Our recent studies demonstrated that ERK1/2 pathway activation triggers the interaction of Shoc2 with the ubiquitin-specific protease USP7. We identified that in the Shoc2 module, USP7 functions as a molecular "switch" that controls the E3 ligase HUWE1 and the HUWE1-induced regulatory feedback loop. We also found that disruption of Shoc2-USP7 binding leads to aberrant activation of the Shoc2-ERK1/2 axis. The zebrafish vertebrate model was then used to show that Shoc2 congenital mutations affecting Shoc2 interaction with USP7 lead to aberrant Shoc2 ubiquitination and signal transmission. Thus, our studies reveal a role for USP7 in the pathogenic mechanisms underlying NSLAH extending our understanding of how ubiquitin-specific proteases regulate intracellular signaling. In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi-protein enzymatic machinery to govern the amplitude of Shoc2-ERK1/2 signals. We also uncover novel molecular mechanisms underlying the pathogenesis of Noonan-like syndrome with loose anagen hair. Overall, these studies significantly advance our understanding of the mechanisms by which non-enzymatic scaffolds regulate the specificity and dynamics of the ERK1/2 signaling networks.
  27. J Biol Chem. 2022 May 04. pii: S0021-9258(22)00456-2. [Epub ahead of print] 102016
      Ubiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like post-translational modification with important biological functions. However, the regulatory mechanisms governing UFM1 modification of target proteins (UFMylation) and the cellular processes controlled by UFMylation remain largely unknown. It has been previously shown that a UFM1-specific protease (UFSP2) mediates the maturation of the UFM1 precursor and drives the de-UFMylation reaction. Furthermore, it has long been thought that UFSP1, an orthologue of UFSP2, is inactive in many organisms, including human, because it lacks an apparent protease domain when translated from the canonical start codon (445AUG). Here, we demonstrate using the combination of site-directed mutagenesis, CRISPR/Cas9 mediated genome editing and mass spectrometry approaches that translation of human UFSP1 initiates from an upstream near-cognate codon, 217CUG, via eukaryotic translation initiation factor eIF2A-mediated translational initiation rather than from the annotated 445AUG, revealing the presence of a catalytic protease domain containing a Cys active site. Moreover, we show that both UFSP1 and UFSP2 mediate maturation of UFM1 and de-UFMylation of target proteins. This study demonstrates that human UFSP1 functions as an active UFM1-specific protease, thus contributing to our understanding of the UFMylation/de-UFMylation process.
    Keywords:  UFM1-specific protease; UFSP1; UFSP2; Ufmylation; eIF2A-mediated translational initiation
  28. FASEB J. 2022 May;36 Suppl 1
      Ubiquitination is a post-translational modification that determines the half-life of many cellular proteins by conjugating a chain of the small protein ubiquitin to a target protein. The ubiquitin chain is then recognized by the 26S proteasome, and subsequently, the ubiquitinated protein is degraded. This ubiquitin-proteosome system (UPS) contains various components that function cooperatively to determine the ubiquitination status of cellular proteins that play key roles in biological events. It has become clear that mis-regulation of protein ubiquitination can lead to diverse human diseases, and multiple UPS components have been identified as promising drug targets. To facilitate studies that define roles of the UPS components in protein ubiquitination, a method that probes the ubiquitination status of the whole proteome in human cells is necessary. Tandem ubiquitin binding entities (TUBEs) is a synthetic protein that binds poly-ubiquitin chains with a dissociation constant in the nanomolar range. Therefore, TUBEs can recognize the poly-ubiquitinated protein and enrich them from human cell lysate. Combining the TUBE-dependent affinity precipitation, stable isotope labeling by amino acids in cell culture (SILAC), and mass spectrometry, we are establishing a method for quantitatively accessing the ubiquitinated proteome in human cells. To preserve the poly-ubiquitinated protein, HEK293 cells were treated with bortezomib, a proteosome inhibitor, to prevent the ubiquitinated proteins from degradation. To perform the affinity precipitation, the recombinant TUBE was conjugated to biotin and immobilized on resin via the Streptavidin-biotin interaction. The lysate of the bortezomib-treated cells was incubated with the immobilized Biotin TUBE to enrich poly-ubiquitinated proteins, which were then digested by the trypsin protease. Following the trypsin digestion, ubiquitinated proteins generated peptides with the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant, which was further immunoprecipitated by antibodies specifically recognizing the K-ɛ-GG remnant and identified by mass spectrometry. To probe proteins with different ubiquitination status under different conditions, SILAC was used to grow one cell sample in normal (light) media and the other cell sample in (heavy) media containing stable isotope labeled amino acids. By comparing the abundance of the light versus heavy peptides from the same ubiquitinated protein, changes in the ubiquitination status of each detected protein can be determined. We anticipate that this method will help profile changes in the ubiquitinated proteome when the UPS components are mutated or interfered by drugs.
  29. Cell Rep. 2022 May 10. pii: S2211-1247(22)00564-2. [Epub ahead of print]39(6): 110797
      The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.
    Keywords:  APEX2; CP: Cell biology; CP: Immunology; ER-mitochondria contact site; HIV-1; TRIM5α; ULK1 complex; autophagy; inflammation; mitochondrial metabolism; proteomics; tripartite motif
  30. FASEB J. 2022 May;36 Suppl 1
      Nucleotide repeat expansions cause multiple neurodegenerative disorders including C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD) and Fragile X-associated tremor/ataxia syndrome (FXTAS). C9 ALS/FTD results from a GGGGCC (G4 C2 ) hexanucleotide repeat expansions within an intron of C9orf72while FXTAS is caused by CGG repeat expansions in the 5'UTR of FMR1. These repeat-containing RNAs elicit toxicity at least in part by triggering repeat-associated non-AUG (RAN) translation, a non-canonical initiation process that generates toxic proteins from GC rich repeats. As repetitive RNA elements form strong RNA secondary structures might impact translational elongation, we explored the impact of ribosome-associated quality control (RQC) pathways on RAN translation. RQC rescues stalled ribosomes and prevents translation of aberrant transcripts, with specific roles for both the mammalian nuclear export mediator factor (NEMF, homologous of Rqc2 in yeast) and the E3 ubiquitin ligase Listerin. Here we show that depletion of NEMF markedly increases the production of RAN products from both G4 C2 and CGG repeats. This effect appears to be mediated post transcriptionally and does not involve the nuclear-cytoplasmic transport functions of the protein. Ongoing studies are characterizing how loss of Listerin impacts RAN translation and repeat RNA stability while exploring whether NEMF meditated CAT-tailing of RAN translation products impacts their turnover and toxicity.
  31. Int J Mol Sci. 2022 Apr 21. pii: 4627. [Epub ahead of print]23(9):
      DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.
    Keywords:  DYT1; ERAD; TorsinA; UBXD1; Ufd1/Npl4; YOD1; dystonia; p97; ubiquitin
  32. FASEB J. 2022 May;36 Suppl 1
      The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR-directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown. We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen-sensitive and castration-resistant PCa cell lines in culture and cell line-derived and patient-derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2. A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR-based phenotypic screens and genome-wide gene expression analyses of wild-type and GCN2-depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2. Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen-sensitive and castration-resistant prostate cancer.
  33. FASEB J. 2022 May;36 Suppl 1
      Autophagy is an essential cellular process by which cellular debris is collected and broken down in the lysosome. Cancer cells rely on autophagy for survival during chemotherapy treatment and periods of hypoxia within solid tumors. In this project, we investigate the ATG13-ATG101 protein complex, a sub-complex of the ULK1 initiatory complex whose regulatory role in autophagy is not completely understood. Our recent data demonstrate that an ULK1-independent ATG13-ATG101 complex is essential for the basal autophagic degradation of protein aggregates-a process known as aggrephagy. Furthermore, we found that the ULK1-independent ATG13-ATG101 complex traffics to condensates of poly-ubiquitinated protein aggregates where it colocalizes with the autophagy regulator ATG9A.1 Our current data suggest that this ATG13-ATG101 complex cooperates with ATG9A in the degradation of these protein aggregates. To further elucidate the function of the ATG13-ATG101 complex, we have developed a protein-fragment complementation assay using a novel split TurboID (BioID) system in which complementary halves of TurboID are fused to ATG13 and ATG101. Using this split BioID system, together with quantitative LC-MS/MS and mutants of ATG13 that cannot bind ULK1, we are identifying interactors that are unique to the ULK1-independent ATG13-ATG101 complex. Based on previous data, we hypothesize that the ATG13-ATG101 sub-complex participates in ATG9A trafficking pathways and recruitment of autophagy regulators to protein aggregate condensates. Thus, our project characterizes the first proximity interactome for the ATG13-ATG101 complex. 1. Kannangara, A. R.; Poole, D. M.; McEwan, C. M.; Youngs, J. C.; Weerasekara, V. K.; Thornock, A. M.; Lazaro, M. T.; Balasooriya, E. R.; Oh, L. M.; Soderblom, E. J.; Lee, J. J.; Simmons, D. L.; Andersen, J. L., BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters. EMBO Rep2021, e51136.
  34. EMBO J. 2022 May 10. e110031
      Autophagy is a cellular degradative pathway that plays diverse roles in maintaining cellular homeostasis. Cellular stress caused by starvation, organelle damage, or proteotoxic aggregates can increase autophagy, which uses the degradative capacity of lysosomal enzymes to mitigate intracellular stresses. Early studies have shown a role for autophagy in the suppression of tumorigenesis. However, work in genetically engineered mouse models and in vitro cell studies have now shown that autophagy can be either cancer-promoting or inhibiting. Here, we summarize the effects of autophagy on cancer initiation, progression, immune infiltration, and metabolism. We also discuss the efforts to pharmacologically target autophagy in the clinic and highlight future areas for exploration.
    Keywords:  ATG; autophagy; cancer; chloroquine; metabolism
  35. bioRxiv. 2021 Sep 16. pii: 2021.09.15.460543. [Epub ahead of print]
      The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) genome is evolving as the viral pandemic continues its active phase around the world. The Papain-like protease (PLpro) is a domain of Nsp3 â€" a large multidomain protein that is an essential component of the replication-transcription complex, making it a good therapeutic target. PLpro is a multi-functional protein encoded in coronaviruses that can cleave viral polyproteins, poly-ubiquitin and protective Interferon Stimulated Gene 15 product, ISG15, which mimics a head-to-tail linked ubiquitin (Ub) dimer. PLpro across coronavirus families showed divergent selectivity for recognition and cleavage of these protein substrates despite sequence conservation. However, it is not clear how sequence changes in SARS-CoV-2 PLpro alter its selectivity for substrates and what outcome this has on the pathogenesis of the virus. We show that SARS-CoV-2 PLpro preferentially binds ISG15 over Ub and K48-linked Ub 2 . We determined crystal structures of PLpro in complex with human K48-Ub 2 and ISG15 revealing that dual domain recognition of ISG15 drives substrate selectivity over Ub and Ub 2 . We also characterized the PLpro substrate interactions using solution NMR, cross-linking mass spectrometry to support that ISG15 is recognized via two domains while Ub 2 binds primarily through one Ub domain. Finally, energetic analysis of the binding interfaces between PLpro from SARS-CoV-1 and SARS-CoV-2 with ISG15 and Ub 2 define the sequence determinants for how PLpros from different coronaviruses recognize two topologically distinct substrates and how evolution of the protease altered its substrate selectivity. Our work reveals how PLpro substrate selectivity may evolve in PLpro coronaviruses variants enabling design of more effective therapeutics.
  36. Commun Biol. 2022 May 11. 5(1): 445
      Effective organization of proteins into functional modules (networks, pathways) requires systems-level coordination between transcription, translation and degradation. Whereas the cooperation between transcription and translation was extensively studied, the cooperative degradation regulation of protein complexes and pathways has not been systematically assessed. Here we comprehensively analyzed degron masking, a major mechanism by which cellular systems coordinate degron recognition and protein degradation. For over 200 substrates with characterized degrons (E3 ligase targeting motifs, ubiquitination sites and disordered proteasomal entry sequences), we demonstrate that degrons extensively overlap with protein-protein interaction sites. Analysis of binding site information and protein abundance comparisons show that regulatory partners effectively outcompete E3 ligases, masking degrons from the ubiquitination machinery. Protein abundance variations between normal and cancer cells highlight the dynamics of degron masking components. Finally, integrative analysis of gene co-expression, half-life correlations and functional relationships between interacting proteins point towards higher-order, co-regulated degradation modules ('degronons') in the proteome.
  37. Nat Commun. 2022 May 12. 13(1): 2640
      The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate. The other structure adopts a helical conformation, displays substrate running in an extended conformation through the pore of the p97 hexamer, and resembles structures reported for other AAA unfoldases. These findings support the model that p97 utilizes a "hand-over-hand" mechanism in which two residues of the substrate are translocated for hydrolysis of two ATPs, one in each of the two p97 AAA ATPase rings. Proteomics analysis supports the model that one p97 complex can bind multiple substrate adaptors or binding partners, and can process substrates with multiple types of ubiquitin modification.
  38. iScience. 2022 May 20. 25(5): 104273
      Neurodegeneration is associated with the aggregation of proteins bearing solvent-exposed hydrophobicity as a result of their misfolding and/or proteolytic cleavage. An understanding of the cellular protein quality control mechanisms which prevent protein aggregation is fundamental to understanding the etiology of neurodegeneration. By examining the metabolism of disease-linked C-terminal fragments of the TAR DNA-binding protein 43 (TDP43), we found that the Bcl-2 associated athanogene 6 (BAG6) functions as a sensor of proteolytic fragments bearing exposed hydrophobicity and prevents their intracellular aggregation. In addition, BAG6 facilitates the ubiquitylation of TDP43 fragments by recruiting the Ub-ligase, Ring finger protein 126 (RNF126). Authenticating its role in preventing aggregation, we found that TDP43 fragments form intracellular aggregates in the absence of BAG6. Finally, we found that BAG6 could interact with and solubilize additional neurodegeneration-associated proteolytic fragments. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.
    Keywords:  Biological sciences; Cell biology; Molecular biology; Molecular neuroscience; Neuroscience
  39. Nucleic Acids Res. 2022 May 12. pii: gkac342. [Epub ahead of print]
      Biomolecular associations forged by specific interaction among structural scaffolds are fundamental to the control and regulation of cell processes. One such structural architecture, characterized by HEAT repeats, is involved in a multitude of cellular processes, including intracellular transport, signaling, and protein synthesis. Here, we review the multitude and versatility of HEAT domains in the regulation of mRNA translation initiation. Structural and cellular biology approaches, as well as several biophysical studies, have revealed that a number of HEAT domain-mediated interactions with a host of protein factors and RNAs coordinate translation initiation. We describe the basic structural architecture of HEAT domains and briefly introduce examples of the cellular processes they dictate, including nuclear transport by importin and RNA degradation. We then focus on proteins in the translation initiation system featuring HEAT domains, specifically the HEAT domains of eIF4G, DAP5, eIF5, and eIF2Bϵ. Comparative analysis of their remarkably versatile interactions, including protein-protein and protein-RNA recognition, reveal the functional importance of flexible regions within these HEAT domains. Here we outline how HEAT domains orchestrate fundamental aspects of translation initiation and highlight open mechanistic questions in the area.
  40. Nature. 2022 May 12.
      SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle (RO) to enable RNA replication1. The SARS-CoV-2 RO is composed of double membrane vesicles (DMVs) tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved are currently unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 RO. NSP3 and NSP4 generate the DMVs while NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6ΔSGF mutant, which arose independently in the α, β, γ, η, ι, and λ variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: to act as a filter in RO-ER communication allowing lipid flow but restricting access of ER luminal proteins to the DMVs, to position and organize DMV clusters, and to mediate contact with lipid droplets (LDs) via the LD-tethering complex DFCP1-Rab18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective track to refurbish them with LD-derived lipids. Importantly, both properly formed NSP6 connectors and LDs are required for SARS-CoV-2 replication. Our findings, uncovering the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, have the potential to fuel the search for broad antiviral agents.