bims-proteo Biomed News
on Proteostasis
Issue of 2021‒06‒27
forty-six papers selected by
Eric Chevet

  1. Trends Cell Biol. 2021 Jun 16. pii: S0962-8924(21)00099-4. [Epub ahead of print]
      Precise distribution of proteins is essential to sustain the viability of cells. A complex network of protein synthesis and targeting factors cooperate with protein quality control systems to ensure protein homeostasis. Defective proteins are inevitably degraded by the ubiquitin-proteasome system and lysosomes. However, due to overlapping targeting information and limited targeting fidelity, certain proteins become mislocalized. In this review, we present the idea that transmembrane dislocases recognize and remove mislocalized membrane proteins from cellular organelles. This enables other targeting attempts and prevents degradation of mislocalized but otherwise functional proteins. These transmembrane dislocases can be found in the outer mitochondrial membrane (OMM) and endoplasmic reticulum (ER). We highlight common principles regarding client recognition and outline open questions in our understanding of transmembrane dislocases.
    Keywords:  AAA-ATPase Msp1/ATAD1/Thorase; ER-associated degradation; P5-type ATPase Spf1/ATP13A1; mitochondrial protein quality control; protein homeostasis; protein quality control
  2. EMBO J. 2021 Jun 21. e107240
      Efficient degradation of by-products of protein biogenesis maintains cellular fitness. Strikingly, the major biosynthetic compartment in eukaryotic cells, the endoplasmic reticulum (ER), lacks degradative machineries. Misfolded proteins in the ER are translocated to the cytosol for proteasomal degradation via ER-associated degradation (ERAD). Alternatively, they are segregated in ER subdomains that are shed from the biosynthetic compartment and are delivered to endolysosomes under control of ER-phagy receptors for ER-to-lysosome-associated degradation (ERLAD). Demannosylation of N-linked oligosaccharides targets terminally misfolded proteins for ERAD. How misfolded proteins are eventually marked for ERLAD is not known. Here, we show for ATZ and mutant Pro-collagen that cycles of de-/re-glucosylation of selected N-glycans and persistent association with Calnexin (CNX) are required and sufficient to mark ERAD-resistant misfolded proteins for FAM134B-driven lysosomal delivery. In summary, we show that mannose and glucose processing of N-glycans are triggering events that target misfolded proteins in the ER to proteasomal (ERAD) and lysosomal (ERLAD) clearance, respectively, regulating protein quality control in eukaryotic cells.
    Keywords:  ER-phagy; ERAD; ERLAD; N-glycan processing; Protein quality control
  3. Biochem Soc Trans. 2021 Jun 22. pii: BST20200381. [Epub ahead of print]
      Molecular chaperones are essential components of the protein quality control system and maintenance of homeostasis. Heat Shock Protein 70 (HSP70), a highly evolutionarily conserved family of chaperones is a key regulator of protein folding, oligomerisation and prevents the aggregation of misfolded proteins. HSP70 chaperone function depends on the so-called 'HSP70-cycle', where HSP70 interacts with and is released from substrates via ATP hydrolysis and the assistance of HSP70 co-factors/co-chaperones, which also provide substrate specificity. The identification of regulatory modules for HSP70 allows the elucidation of HSP70 specificity and target selectivity. Here, we discuss how the HSP70 cycle is functionally linked with the cycle of the Ubiquitin-like molecule NEDD8. Using as an example the DNA damage response, we present a model where HSP70 acts as a sensor of the NEDD8 cycle. The NEDD8 cycle acts as a regulatory module of HSP70 activity, where conversion of poly-NEDD8 chains into mono-NEDD8 upon DNA damage activates HSP70, facilitating the formation of the apoptosome and apoptosis execution.
    Keywords:  DNA damage response; NEDD8; NEDD8 chains; NEDP1; heat shock proteins
  4. Annu Rev Biochem. 2021 Jun 20. 90 659-679
      The polytopic, endoplasmic reticulum (ER) membrane protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, the key intermediate in the synthesis of cholesterol and many nonsterol isoprenoids including geranylgeranyl pyrophosphate (GGpp). Transcriptional, translational, and posttranslational feedback mechanisms converge on this reductase to ensure cells maintain a sufficient supply of essential nonsterol isoprenoids but avoid overaccumulation of cholesterol and other sterols. The focus of this review is mechanisms for the posttranslational regulation of HMG CoA reductase, which include sterol-accelerated ubiquitination and ER-associated degradation (ERAD) that is augmented by GGpp. We discuss how GGpp-induced ER-to-Golgi trafficking of the vitamin K2 synthetic enzyme UbiA prenyltransferase domain-containing protein-1 (UBIAD1) modulates HMG CoA reductase ERAD to balance the synthesis of sterol and nonsterol isoprenoids. We also summarize the characterization of genetically manipulated mice, which established that sterol-accelerated, UBIAD1-modulated ERAD plays a major role in regulation of HMG CoA reductase and cholesterol metabolism in vivo.
    Keywords:  ER-associated degradation; Golgi; cholesterol; geranylgeranyl pyrophosphate; isoprenoid; ubiquitin
  5. EMBO J. 2021 Jun 21. e100715
      Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.
    Keywords:  Parkinson’s disease; Rhot1; Rhot2; eIF2α; megamitochondria
  6. Autophagy. 2021 Jun 24. 1-2
      Cellular stress response mechanisms typically increase organellar quantity and volume. To restore cellular homeostasis and organellar integrity, the surplus organelles are cleared by macroautophagy/autophagy, an intracellular process that shuttles cytoplasmic material to the lysosomes for degradation. The degradation is mediated by autophagy receptors that selectively link the degradable cargo to the autophagy machinery. Studies have identified receptors for the degradation of mitochondria, endoplasmic reticulum, lysosomes, and peroxisomes. The autophagic degradation of the Golgi, named Golgiphagy, however, has remained undefined. The Golgi is essential for the processing, sorting and trafficking of proteins and lipids in the secretory pathway. In a recent study, we identified CALCOCO1 as a Golgiphagy receptor in response to nutrient deprivation. CALCOCO1 interacts with Golgi membranes by binding to cytoplasmic Ankyrin repeat (AR) domains of Golgi resident ZDHHC17 and ZDHHC13 palmitoyltransferases (PATs) via a defined zDHHC-AR-binding motif (zDABM) to recruit autophagy machinery. Lack of CALCOCO1 in cells causes an impaired Golgiphagy and expansion of the Golgi.
    Keywords:  Autophagy receptor; CALCOCO1; Golgi; Golgiphagy; Golgiphagy receptor; ZDHHC17; zDABM motif
  7. Hepatology. 2021 Jun 25.
      BACKGROUND & AIMS: The unfolded protein response (UPR) is a coordinated cellular response to endoplasmic reticulum (ER) stress that functions to maintain cellular homeostasis. When ER stress is unresolved, the UPR can trigger apoptosis. Pathways within the UPR influence bile acid metabolism in adult animal models and adult human liver diseases, however the UPR has not been studied in young animal models or pediatric liver diseases. In this study we sought to determine if weanling age mice had altered UPR activation compared to adult mice which could lead to increased bile acid induced hepatic injury APPROACH & RESULTS: We demonstrate that after 7 days of cholic acid (CA) feeding to wild type (WT) animals, weanling age mice have a 2-fold greater serum ALT levels compared to adult mice, with increased hepatic apoptosis. Weanling mice fed CA have increased hepatic nuclear X-box binding protein 1 spliced (XBP1s) expression, but cannot increase expression of its protective downstream targets endoplasmic reticulum DNA J domain-containing protein 4 (ERdj4) and ER degradation enhancing α-mannoside (EDEM). In response to tunicamycin induced ER stress, young mice have blunted expression of several UPR pathways compared to adult mice. CA feeding to adult liver-specific XBP1 knockout (LS-XBP1-/- ) mice, which are unable to resolve hepatic ER stress, leads to increased serum ALT and C/EBP homologous protein (CHOP), a proapoptotic UPR molecule, expression to levels similar to CA fed LS-XBP1-/- weanlings.CONCLUSIONS: Weanling mice have attenuated hepatic XBP1 signaling and impaired UPR activation with resultant increased susceptibility to bile acid induced injury.
    Keywords:  ER stress; bile acid; cholestasis; endoplasmic reticulum; liver
  8. Annu Rev Cell Dev Biol. 2021 Jun 21.
      Selective autophagy is the lysosomal degradation of specific intracellular components sequestered into autophagosomes, late endosomes, or lysosomes through the activity of selective autophagy receptors (SARs). SARs interact with autophagy-related (ATG)8 family proteins via sequence motifs called LC3-interacting region (LIR) motifs in vertebrates and Atg8-interacting motifs (AIMs) in yeast and plants. SARs can be divided into two broad groups: soluble or membrane bound. Cargo or substrate selection may be independent or dependent of ubiquitin labeling of the cargo. In this review, we discuss mechanisms of mammalian selective autophagy with a focus on the unifying principles employed in substrate recognition, interaction with the forming autophagosome via LIR-ATG8 interactions, and the recruitment of core autophagy components for efficient autophagosome formation on the substrate. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see for revised estimates.
  9. Mol Biol Cell. 2021 Jun 23. mbcE20110747
      A network of chaperones and ubiquitin ligases sustain intracellular proteostasis, and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases: Spinocerebellar ataxia Type 3 (SCA3) and Huntington's disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain is diminished when polyQ repeat proteins (Ataxin-3/Huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyglutamine aggregates.
  10. Biochim Biophys Acta Mol Cell Res. 2021 Jun 17. pii: S0167-4889(21)00135-X. [Epub ahead of print] 119081
      The DYRK (Dual-specificity tYrosine-phosphorylation Regulated protein Kinase) family consists of five related protein kinases (DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4). DYRKs show homology to Drosophila Minibrain, and DYRK1A in human chromosome 21 is responsible for various neuronal disorders including human Down syndrome. Here we report identification of cellular proteins that associate with specific members of DYRKs. Cellular proteins with molecular masses of 90, 70, and 50-kDa associated with DYRK1B and DYRK4. These proteins were identified as molecular chaperones Hsp90, Hsp70, and Cdc37, respectively. Microscopic analysis of GFP-DYRKs showed that DYRK1A and DYRK1B were nuclear, while DYRK2, DYRK3, and DYRK4 were mostly cytoplasmic in COS7 cells. Overexpression of DYRK1B induced nuclear re-localization of these chaperones with DYRK1B. Treatment of cells with specific Hsp90 inhibitors, geldanamycin and 17-AAG, abolished the association of Hsp90 and Cdc37 with DYRK1B and DYRK4, but not of Hsp70. Inhibition of Hsp90 chaperone activity affected intracellular dynamics of DYRK1B and DYRK4. DYRK1B and DYRK4 underwent rapid formation of cytoplasmic punctate dots after the geldanamycin treatment, suggesting that the chaperone function of Hsp90 is required for prevention of protein aggregation of the target kinases. Prolonged inhibition of Hsp90 by geldanamycin, 17-AAG, or ganetespib, decreased cellular levels of DYRK1B and DYRK4. Finally, DYRK1B and DYRK4 were ubiquitinated in cells, and ubiquitinated DYRK1B and DYRK4 further increased by Hsp90 inhibition with geldanamycin. Taken together, these results indicate that Hsp90 and Cdc37 discriminate specific members of the DYRK kinase family and play an important role in quality control of these client kinases in cells.
    Keywords:  Cdc37; DYRK; Hsp90; Molecular chaperone; Protein kinase; Protein quality control
  11. Front Cell Dev Biol. 2021 ;9 684885
      p62/SQSTM1 (sequestosome-1) is a key protein involved in multiple cellular bioprocesses including autophagy, nutrient sensing, cell growth, cell death, and survival. Therefore, it is implicated in human diseases such as obesity and cancer. Here, we show that the CUL5-ASB6 complex is a ubiquitin E3 ligase complex mediating p62 ubiquitination and degradation. Depletion of CUL5 or ASB6 induced p62 accumulation, and overexpression of ASB6 promoted ubiquitination and degradation of p62. Functionally, ASB6 overexpression can inhibit the proliferation of MEF and hepatocellular carcinoma cells by reducing p62 protein level, and impair the occurrence of autophagy. Overall, our study identified a new molecular mechanism regulating p62 stability, which may provide additional insights for understanding the delicate control of p62 and cell proliferation-autophagy control in physiological and pathological settings.
    Keywords:  ASB6; CUL5; autophagy; p62; proliferation; ubiquitination
  12. JHEP Rep. 2021 Aug;3(4): 100297
      Background & Aims: A single point mutation in the Z-variant of alpha 1-antitrypsin (Z-AAT) alone can lead to both a protein folding and trafficking defect, preventing its exit from the endoplasmic reticulum (ER), and the formation of aggregates that are retained as inclusions within the ER of hepatocytes. These defects result in a systemic AAT deficiency (AATD) that causes lung disease, whereas the ER-retained aggregates can induce severe liver injury in patients with ZZ-AATD. Unfortunately, therapeutic approaches are still limited and liver transplantation represents the only curative treatment option. To overcome this limitation, a better understanding of the molecular basis of ER aggregate formation could provide new strategies for therapeutic intervention.Methods: Our functional and omics approaches here based on human hepatocytes from patients with ZZ-AATD have enabled the identification and characterisation of the role of the protein disulfide isomerase (PDI) A4/ERP72 in features of AATD-mediated liver disease.
    Results: We report that 4 members of the PDI family (PDIA4, PDIA3, P4HB, and TXNDC5) are specifically upregulated in ZZ-AATD liver samples from adult patients. Furthermore, we show that only PDIA4 knockdown or alteration of its activity by cysteamine treatment can promote Z-AAT secretion and lead to a marked decrease in Z aggregates. Finally, detailed analysis of the Z-AAT interactome shows that PDIA4 silencing provides a more conducive environment for folding of the Z mutant, accompanied by reduction of Z-AAT-mediated oxidative stress, a feature of AATD-mediated liver disease.
    Conclusions: PDIA4 is involved in AATD-mediated liver disease and thus represents a therapeutic target for inhibition by drugs such as cysteamine. PDI inhibition therefore represents a potential therapeutic approach for treatment of AATD.
    Lay summary: Protein disulfide isomerase (PDI) family members, and particularly PDIA4, are upregulated and involved in alpha 1-antitrypsin deficiency (AATD)-mediated liver disease in adults. PDI inhibition upon cysteamine treatment leads to improvements in features of AATD and hence represents a therapeutic approach for treatment of AATD-mediated liver disease.
    Keywords:  AAT, alpha 1-antitrypsin; AATD, alpha 1-antitrypsin deficiency; Alpha 1-antitrypsin deficiency; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; Cysteamine; ER, endoplasmic reticulum; FFPE, formalin-fixed paraffin-embedded; FKBP10, FK506-binding protein (FKBP) isoform 10; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; IP, immunoprecipitation; Liver damage; NHK, null Hong Kong variant of AAT; P4HB, prolyl 4-hydroxylase subunit beta/PDIA1; PDI, protein disulfide isomerase; PDIA3, protein disulfide isomerase family A member 3/ERP57; PDIA4; PDIA4, protein disulfide isomerase family A member 4/ERP70/ERP72; PDIi, PDI inhibitors; Protein disulfide isomerase; ROS, reactive oxygen species; SURF4, proteins Surfeit 4; Scr, scramble; TRX, thioredoxin; TXNDC5, thioredoxin domain containing 5/PDIA15; Treatment; WT, wild-type; Z-AAT, alpha 1-antitrypsin Z variant; ZZ, homozygosis for the Z mutant allele; siRNA, small RNA interference; ΔF508-CFTR, most common mutation of CFTR, which deletes phenylalanine508
  13. Sci Adv. 2021 Jun;pii: eabg3012. [Epub ahead of print]7(26):
      Protein aggregation causes intracellular changes in neurons, which elicit signals to modulate proteostasis in the periphery. Beyond the nervous system, a fundamental question is whether other organs also communicate their proteostasis status to distal tissues. Here, we examine whether proteostasis of the germ line influences somatic tissues. To this end, we induce aggregation of germline-specific PGL-1 protein in germline stem cells of Caenorhabditis elegans Besides altering the intracellular mitochondrial network of germline cells, PGL-1 aggregation also reduces the mitochondrial content of somatic tissues through long-range Wnt signaling pathway. This process induces the unfolded protein response of the mitochondria in the soma, promoting somatic mitochondrial fragmentation and aggregation of proteins linked with neurodegenerative diseases such as Huntington's and amyotrophic lateral sclerosis. Thus, the proteostasis status of germline stem cells coordinates mitochondrial networks and protein aggregation through the organism.
  14. Cell Death Differ. 2021 Jun 22.
      During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.
  15. Nat Commun. 2021 06 24. 12(1): 3928
      The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1-/- mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart.
  16. Cell Chem Biol. 2021 Jun 12. pii: S2451-9456(21)00261-0. [Epub ahead of print]
      Hydrogen sulfide (H2S) is a gasotransmitter with broad physiological activities, including protecting cells against stress, but little is known about the regulation of cellular H2S homeostasis. We have performed a high-content small-molecule screen and identified genotoxic agents, including cancer chemotherapy drugs, as activators of intracellular H2S levels. DNA damage-induced H2S in vitro and in vivo. Mechanistically, DNA damage elevated autophagy and upregulated H2S-generating enzyme CGL; chemical or genetic disruption of autophagy or CGL impaired H2S induction. Importantly, exogenous H2S partially rescued autophagy-deficient cells from genotoxic stress. Furthermore, stressors that are not primarily genotoxic (growth factor depletion and mitochondrial uncoupler FCCP) increased intracellular H2S in an autophagy-dependent manner. Our findings highlight the role of autophagy in H2S production and suggest that H2S generation may be a common adaptive response to DNA damage and other stressors.
    Keywords:  CGL enzyme; DNA damage response; P3 probe; SF7-AM probe; autophagy; cancer chemotherapy; high-throughput screening; hydrogen sulfide; stress response; sulfide metabolism
  17. J Mol Biol. 2021 Jun 18. pii: S0022-2836(21)00333-8. [Epub ahead of print] 167109
      Secretory and membrane proteins follow either the signal recognition particle (SRP)-dependent cotranslational translocation pathway or the SRP-independent Sec62/Sec63-dependent posttranslational pathway for their translocation across the endoplasmic reticulum (ER). However, increasing evidence suggests that most proteins are cotranslationally targeted to the ER, suggesting mixed mechanisms. It remains unclear how these two pathways cooperate. Previous studies have shown that Spc3, a signal-anchored protein, requires SRP and Sec62 for its biogenesis. This study investigated the targeting and topogenesis of Spc3 and the step at which SRP and Sec62 act using in vivo and in vitro translocation assays and co-immunoprecipitation. Our data suggest that Spc3 reaches its final topology in two steps: it enters the ER lumen head-first and then inverts its orientation. The first step is partially dependent on SRP, although independent of the Sec62/Sec63 complex. The second step is mediated by the Sec62/Sec63 complex. These data suggest that SRP and Sec62 act on a distinct step in the topogenesis of Spc3.
    Keywords:  Sec61; Sec62; membrane protein; topology; yeast
  18. J Cell Biol. 2021 Sep 06. pii: e202010177. [Epub ahead of print]220(9):
      The Hedgehog pathway, critical to vertebrate development, is organized in primary cilia. Activation of signaling causes the Hedgehog receptor Ptch1 to exit cilia, allowing a second receptor, Smo, to accumulate in cilia and activate the downstream steps of the pathway. Mechanisms regulating the dynamics of these receptors are unknown, but the ubiquitination of Smo regulates its interaction with the intraflagellar transport system to control ciliary levels. A focused screen of ubiquitin-related genes identified nine required for maintaining low ciliary Smo at the basal state. These included cytoplasmic E3s (Arih2, Mgrn1, and Maea), a ciliary localized E3 (Wwp1), a ciliary localized E2 (Ube2l3), a deubiquitinase (Bap1), and three adaptors (Kctd5, Skp1a, and Skp2). The ciliary E3, Wwp1, binds Ptch1 and localizes to cilia at the basal state. Activation of signaling removes both Ptch1 and Wwp1 from cilia, thus providing an elegant mechanism for Ptch1 to regulate ciliary Smo levels.
  19. Dev Cell. 2021 Jun 24. pii: S1534-5807(21)00481-0. [Epub ahead of print]
      Mitochondria are critical metabolic and signaling hubs, and dysregulated mitochondrial homeostasis is implicated in many diseases. Degradation of damaged mitochondria by selective GABARAP/LC3-dependent macro-autophagy (mitophagy) is critical for maintaining mitochondrial homeostasis. To identify alternate forms of mitochondrial quality control that functionally compensate if mitophagy is inactive, we selected for autophagy-dependent cancer cells that survived loss of LC3-dependent autophagosome formation caused by inactivation of ATG7 or RB1CC1/FIP200. We discovered rare surviving autophagy-deficient clones that adapted to maintain mitochondrial homeostasis after gene inactivation and identified two enhanced mechanisms affecting mitochondria including mitochondrial dynamics and mitochondrial-derived vesicles (MDVs). To further understand these mechanisms, we quantified MDVs via flow cytometry and confirmed an SNX9-mediated mechanism necessary for flux of MDVs to lysosomes. We show that the autophagy-dependent cells acquire unique dependencies on these processes, indicating that these alternate forms of mitochondrial homeostasis compensate for loss of autophagy to maintain mitochondrial health.
    Keywords:  ATG7; FIP200; SNX9; autophagy; cancer; late endosomes; mitochondria; mitochondrial dynamics; mitochondrial-derived vesicles; mitophagy
  20. Nucleic Acids Res. 2021 Jun 22. pii: gkab511. [Epub ahead of print]
      Rad51 is the key protein in homologous recombination that plays important roles during DNA replication and repair. Auxiliary factors regulate Rad51 activity to facilitate productive recombination, and prevent inappropriate, untimely or excessive events, which could lead to genome instability. Previous genetic analyses identified a function for Rrp1 (a member of the Rad5/16-like group of SWI2/SNF2 translocases) in modulating Rad51 function, shared with the Rad51 mediator Swi5-Sfr1 and the Srs2 anti-recombinase. Here, we show that Rrp1 overproduction alleviates the toxicity associated with excessive Rad51 levels in a manner dependent on Rrp1 ATPase domain. Purified Rrp1 binds to DNA and has a DNA-dependent ATPase activity. Importantly, Rrp1 directly interacts with Rad51 and removes it from double-stranded DNA, confirming that Rrp1 is a translocase capable of modulating Rad51 function. Rrp1 affects Rad51 binding at centromeres. Additionally, we demonstrate in vivo and in vitro that Rrp1 possesses E3 ubiquitin ligase activity with Rad51 as a substrate, suggesting that Rrp1 regulates Rad51 in a multi-tiered fashion.
  21. Curr Biol. 2021 Jun 15. pii: S0960-9822(21)00750-8. [Epub ahead of print]
      Macroautophagy (hereafter referred to as autophagy) is a conserved process that promotes cellular homeostasis through the degradation of cytosolic components, also known as cargo. During autophagy, cargo is sequestered into double-membrane vesicles called autophagosomes, which are predominantly transported in the retrograde direction to the perinuclear region to fuse with lysosomes, thus ensuring cargo degradation.1 The mechanisms regulating directional autophagosomal transport remain unclear. The ATG8 family of proteins associates with autophagosome membranes2 and plays key roles in autophagy, including the movement of autophagosomes. This is achieved via the association of ATG8 with adaptor proteins like FYCO1, involved in the anterograde transport of autophagosomes toward the cell periphery.1,3-5 We previously reported that phosphorylation of LC3B/ATG8 on threonine 50 (LC3B-T50) by the Hippo kinase STK4/MST1 is required for autophagy through unknown mechanisms.6 Here, we show that STK4-mediated phosphorylation of LC3B-T50 reduces the binding of FYCO1 to LC3B. In turn, impairment of LC3B-T50 phosphorylation decreases starvation-induced perinuclear positioning of autophagosomes as well as their colocalization with lysosomes. Moreover, a significantly higher number of LC3B-T50A-positive autophagosomes undergo aberrant anterograde movement to axonal tips in mammalian neurons and toward the periphery of mammalian cells. Our data support a role of a nutrient-sensitive STK4-LC3B-FYCO1 axis in the regulation of the directional transport of autophagosomes, a key step of the autophagy process, via the post-translational modification of LC3B.
    Keywords:  FYCO1; Hippo kinases; LC3B; STK4; autophagy; starvation; trafficking; vesicle transport
  22. J Biol Chem. 2021 Jun 16. pii: S0021-9258(21)00686-4. [Epub ahead of print] 100886
      The aryl hydrocarbon receptor (AHR) is a transcription factor activated by exogenous halogenated polycyclic aromatic hydrocarbon compounds, including the environmental toxin TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and naturally occurring dietary and endogenous compounds. Activated AHR enhances transcription of specific genes including phase I and phase II metabolism enzymes and other targets genes like the TCDD-inducible poly ADP-ribose Polymerase (TiPARP). The regulation of AHR activation is a dynamic process: immediately following transcriptional activation of the AHR by TCDD, AHR is exported from the nucleus to the cytoplasm where it is subjected to proteasomal degradation. However, the mechanisms regulating AHR degradation are not well understood. Here we studied the role of two enzymes reported to enhance AHR breakdown: the Cullin 4B (CUL4B)AHR complex, an E3 ubiquitin ligase that targets the AHR and other proteins for ubiquitination, and TiPARP, which targets proteins for ADP-ribosylation, a posttranslational modification that can increase susceptibility to degradation. Using a wild type mouse embryonic fibroblast (MEF) cell line and a MEF cell line in which CUL4B has been deleted (MEFCul4b-null), we discovered that loss of CUL4B partially prevented AHR degradation following TCDD exposure, while knocking down TiPARP in MEFCul4b-null cells completely abolished AHR degradation upon TCDD treatment. Increased TCDD-activated AHR protein levels in MEFCul4b-null and in MEFCul4b-null cells in which TiPARP was knocked down led to enhanced AHR transcriptional activity, indicating that CUL4B and TiPARP restrain AHR action. This study reveals a novel function of TiPARP in controlling TCDD-activated AHR nuclear export and subsequent proteasomal degradation.
    Keywords:  2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin); E3 ubiquitin ligase; TCDD-inducible poly(ADP-ribose) polymerase (TiPARP, PARP7, ARTD14); aryl hydrocarbon receptor (AHR); cullin 4B (CUL4B); cytochrome P450 (CYP450); interferon beta 1 (Ifnb1); mouse embryonic fibroblasts (MEFs); nuclear export; proteasome; protein degradation
  23. J Biol Chem. 2021 Jun 17. pii: S0021-9258(21)00691-8. [Epub ahead of print] 100891
      Regulation of cellular proliferation and quiescence is a central issue in biology that has been studied using model unicellular eukaryotes, such as the fission yeast Schizosaccharomyces pombe. We previously reported that the ubiquitin/proteasome pathway and autophagy are essential to maintain quiescence induced by nitrogen deprivation in S. pombe; however, specific ubiquitin ligases that maintain quiescence are not fully understood. Here we investigated the SPX-RING type ubiquitin ligase Pqr1, identified as required for quiescence in a genetic screen. Pqr1 is found to be crucial for vacuolar proteolysis, the final step of autophagy, through proper regulation of phosphate and its polymer polyphosphate. Pqr1 restricts phosphate uptake into the cell through ubiquitination and subsequent degradation of phosphate transporters on plasma membranes. We hypothesized that Pqr1 may act as the central regulator for phosphate control in S. pombe, through the function of the SPX domain involved in phosphate sensing. Deletion of pqr1+ resulted in hyper-accumulation of intracellular phosphate and polyphosphate, and in improper autophagy-dependent proteolysis under conditions of nitrogen starvation. Polyphosphate hyper-accumulation in pqr1+-deficient cells was mediated by the polyphosphate synthase VTC complex in vacuoles. Simultaneous deletion of VTC complex subunits rescued Pqr1 mutant phenotypes, including defects in proteolysis and loss of viability during quiescence. We conclude that excess polyphosphate may interfere with proteolysis in vacuoles by mechanisms that as yet remain unknown. The present results demonstrate a connection between polyphosphate metabolism and vacuolar functions for proper autophagy-dependent proteolysis, and we propose that polyphosphate homeostasis contributes to maintenance of cellular viability during quiescence.
    Keywords:  autophagy; cell cycle; fission yeast; life span; polyphosphate; vacuole
  24. Sci Adv. 2021 Jun;pii: eabg2517. [Epub ahead of print]7(26):
      Many intracellular pathogens avoid detection by their host cells. However, it remains unknown how they avoid being tagged by ubiquitin, an initial step leading to antimicrobial autophagy. Here, we show that the intracellular bacterial pathogen Rickettsia parkeri uses two protein-lysine methyltransferases (PKMTs) to modify outer membrane proteins (OMPs) and prevent their ubiquitylation. Mutants deficient in the PKMTs were avirulent in mice and failed to grow in macrophages because of ubiquitylation and autophagic targeting. Lysine methylation protected the abundant surface protein OmpB from ubiquitin-dependent depletion from the bacterial surface. Analysis of the lysine-methylome revealed that PKMTs modify a subset of OMPs, including OmpB, by methylation at the same sites that are modified by host ubiquitin. These findings show that lysine methylation is an essential determinant of rickettsial pathogenesis that shields bacterial proteins from ubiquitylation to evade autophagic targeting.
  25. EMBO J. 2021 Jun 22. e108050
      Selective autophagy mediates specific degradation of unwanted cytoplasmic components to maintain cellular homeostasis. The suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6)-formed bodies (SGS3/RDR6 bodies) are essential for siRNA amplification in planta. However, whether autophagy receptors regulate selective turnover of SGS3/RDR6 bodies is unknown. By analyzing the transcriptomic response to virus infection in Arabidopsis, we identified a virus-induced small peptide 1 (VISP1) composed of 71 amino acids, which harbor a ubiquitin-interacting motif that mediates interaction with autophagy-related protein 8. Overexpression of VISP1 induced selective autophagy and compromised antiviral immunity by inhibiting SGS3/RDR6-dependent viral siRNA amplification, whereas visp1 mutants exhibited opposite effects. Biochemistry assays demonstrate that VISP1 interacted with SGS3 and mediated autophagic degradation of SGS3/RDR6 bodies. Further analyses revealed that overexpression of VISP1, mimicking the sgs3 mutant, impaired biogenesis of endogenous trans-acting siRNAs and up-regulated their targets. Collectively, we propose that VISP1 is a small peptide receptor functioning in the crosstalk between selective autophagy and RNA silencing.
    Keywords:  RNA silencing; SGS3; autophagy; peptide; plant virus
  26. Science. 2021 06 11. 372(6547): 1215-1219
      Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.
  27. Front Mol Biosci. 2021 ;8 694012
      The ATP-dependent Hsp70s are evolutionary conserved molecular chaperones that constitute central hubs of the cellular protein quality surveillance network. None of the other main chaperone families (Tig, GroELS, HtpG, IbpA/B, ClpB) have been assigned with a comparable range of functions. Through a multitude of functions Hsp70s are involved in many cellular control circuits for maintaining protein homeostasis and have been recognized as key factors for cell survival. Three mechanistic properties of Hsp70s are the basis for their high versatility. First, Hsp70s bind to short degenerate sequence motifs within their client proteins. Second, Hsp70 chaperones switch in a nucleotide-controlled manner between a state of low affinity for client proteins and a state of high affinity for clients. Third, Hsp70s are targeted to their clients by a large number of cochaperones of the J-domain protein (JDP) family and the lifetime of the Hsp70-client complex is regulated by nucleotide exchange factors (NEF). In this review I will discuss advances in the understanding of the molecular mechanism of the Hsp70 chaperone machinery focusing mostly on the bacterial Hsp70 DnaK and will compare the two other prokaryotic Hsp70s HscA and HscC with DnaK.
    Keywords:  HscA; HscC; Hsp70; allostery; molecular chaperone; protein folding; stress response
  28. Front Microbiol. 2021 ;12 647410
      Enteroviruses (EVs) usurp the host autophagy pathway for pro-viral functions; however, the consequence of EV-induced diversion of autophagy on organelle quality control is poorly defined. Using coxsackievirus B3 (CVB3) as a model EV, we explored the interplay between EV infection and selective autophagy receptors, i.e., Tax1-binding protein 1/TRAF6-binding protein (T6BP), optineurin (OPTN), and nuclear dot 10 protein 52 (NDP52), known to be involved in regulating the clearance of damaged mitochondria, a process termed as mitophagy. Following CVB3 infection, we showed significant perturbations of the mitochondrial network coincident with degradation of the autophagy receptor protein T6BP, similar phenomenon to what we previously observed on NDP52. Notably, protein levels of OPTN are not altered during early infection and slightly reduced upon late infection. Cell culture studies revealed that T6BP degradation occurs independent of the function of host caspases and viral proteinase 3C, but requires the proteolytic activity of viral proteinase 2A. Further investigation identified the cleavage site on T6BP after the amino acid 621 that separates the C-terminal ubiquitin-binding domain from the other functional domains at the N-terminus. Genetic silencing of T6BP and OPTN results in the attenuation of CVB3 replication, suggesting a pro-viral activity for these two proteins. Finally, functional assessment of cleaved fragments from NDP52 and T6BP revealed abnormal binding affinity and impaired capacity to be recruited to depolarized mitochondria. Collectively, these results suggest that CVB3 targets autophagy receptors to impair selective autophagy.
    Keywords:  TRAF6-binding protein; Tax1-binding protein 1; calcium-binding and coiled-coil domain-containing protein 2/nuclear dot 10 protein 52; coxsackievirus; enterovirus; optineurin; selective autophagy
  29. J Cell Biol. 2021 Aug 02. pii: e202102070. [Epub ahead of print]220(8):
      Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.
  30. Nucleic Acids Res. 2021 Jun 22. pii: gkab532. [Epub ahead of print]
      Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3'CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1's accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
  31. Mol Biol Cell. 2021 Jun 23. mbcE21040169
      The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text].
  32. J Immunol. 2021 Jun 23. pii: ji2100299. [Epub ahead of print]
      Receptor-interacting protein kinase-1 (RIPK1) is a master regulator of the TNF-α-induced cell death program. The function of RIPK1 is tightly controlled by posttranslational modifications, including linear ubiquitin chain assembly complex-mediated linear ubiquitination. However, the physiological function and molecular mechanism by which linear ubiquitination of RIPK1 regulates TNF-α-induced intracellular signaling remain unclear. In this article, we identified Lys627 residue as a major linear ubiquitination site in human RIPK1 (or Lys612 in murine RIPK1) and generated Ripk1K612R/K612R mice, which spontaneously develop systemic inflammation triggered by sustained emergency hematopoiesis. Mechanistically, without affecting NF-κB activation, Ripk1K612R/K612R mutation enhances apoptosis and necroptosis activation and promotes TNF-α-induced cell death. The systemic inflammation and hematopoietic disorders in Ripk1K612R/K612R mice are completely abolished by deleting TNF receptor 1 or both RIPK3 and Caspase-8. These data suggest the critical role of TNF-α-induced cell death in the resulting phenotype in Ripk1K612R/K612R mice. Together, our results demonstrate that linear ubiquitination of RIPK1 on K612 is essential for limiting TNF-α-induced cell death to further prevent systemic inflammation.
  33. Autophagy. 2021 Jun 21. 1-17
      Macroautophagy/autophagy-related proteins regulate infectious and inflammatory diseases in autophagy-dependent or -independent manner. However, the role of a newly identified mammalian-specific autophagy protein-BECN2 (beclin 2) in innate immune regulation is largely unknown. Here we showed that loss of BECN2 enhanced the activities of NLRP3, AIM2, NLRP1, and NLRC4 inflammasomes upon ligand stimulations. Mechanistically, BECN2 interacted with inflammasome sensors and mediated their degradation through a ULK1- and ATG9A-dependent, but BECN1-WIPI2-ATG16L1-LC3-independent, non-canonical autophagic pathway. BECN2 recruited inflammasome sensors on ATG9A+ vesicles to form a complex (BECN2-ATG9A-sensors) upon ULK1 activation. Three soluble NSF attachment protein receptor (SNARE) proteins (SEC22A, STX5, and STX6) were further shown to mediate the BECN2-ATG9A-dependent inflammasome sensor degradation. Loss of BECN2 promoted alum-induced peritonitis, which could be rescued by the ablation of CASP1 in Becn2-deficient mice. Hence, BECN2 negatively regulated inflammasome activation to control inflammation, serving as a potential therapeutic target for the treatment of infectious and inflammatory diseases.Abbreviations: AIM2: absent in melanoma 2; ATG: autophagy related; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cells; BMDM: bone marrow-derived macrophages; CASP1: caspase 1; CQ: chloroquine; gMDSC: granulocytic myeloid-derived suppressor cells; IL: interleukin; LPS: lipopolysaccharide; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; mMDSC: monocytic myeloid-derived suppressor cells; NLRC4: NLR family CARD domain containing 4; NLRP1: NLR family pyrin domain containing 1; NLRP3: NLR family pyrin domain containing 3; PECs: peritoneal exudate cells; PYCARD/ASC: apoptosis-associated speck-like protein containing a caspase activation and recruitment domain; SNAREs: soluble NSF attachment protein receptors; STX5: syntaxin 5; STX6: syntaxin 6; ULK1: unc-51 like autophagy activating kinase 1; WIPI: WD repeat domain, phosphoinositide interacting.
    Keywords:  ATG9A; Alum-induced peritonitis; BECN2; STX5-STX6-SEC22A-mediated membrane fusion; inflammasome; non-canonical autophagy
  34. J Genet Genomics. 2021 May 21. pii: S1673-8527(21)00102-8. [Epub ahead of print]
      The UFMylation modification is a novel ubiquitin-like conjugation system, consisting of UBA5 (E1), UFC1 (E2), UFL1 (E3), and the conjugating molecule UFM1. Deficiency in this modification leads to embryonic lethality in mice and diseases in humans. However, the function of UFL1 is poorly characterized. Studies on Ufl1 conditional knockout mice have demonstrated that the deletion of Ufl1 in cardiomyocytes and in intestinal epithelial cells causes heart failure and increases susceptibility to experimentally induced colitis, respectively, suggesting an essential role of UFL1 in the maintenance of the homeostasis in these organs. Yet, its physiological function in other tissues and organs remains completely unknown. In this study, we generate the nephron tubules specific Ufl1 knockout mice and find that the absence of Ufl1 in renal tubular results in kidney atrophy and interstitial fibrosis. In addition, Ufl1 deficiency causes the activation of unfolded protein response and cell apoptosis, which may be responsible for the kidney atrophy and interstitial fibrosis. Collectively, our results have demonstrated the crucial role of UFL1 in regulating kidney function and maintenance of endoplasmic reticulum homoeostasis, providing another layer of understanding kidney atrophy.
    Keywords:  ER stress–induced apoptosis; Kidney atrophy; UFMylation modification; UPR-PERK signaling pathway; Ufl1; Ufl1(fl/fl)PAX8(Cre/+) mice
  35. Nucleic Acids Res. 2021 Jun 23. pii: gkab522. [Epub ahead of print]
      Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.
  36. J Biol Chem. 2021 Jun 19. pii: S0021-9258(21)00700-6. [Epub ahead of print] 100900
      Immune-stimulatory ligands, such as major histocompatibility complex (MHC) molecules and the T-cell costimulatory ligand CD86 are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, while virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from non-substrates are poorly understood. Here we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi's sarcoma herpesvirus (KSHV) modulator of immune recognition (MIR)2, using deep mutational scanning (DMS). We identified a highly specific recognition surface in the hydrophobic core of the CD86 TM domain that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TM domain, but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, while MIR2 recognized CD86 with a TM domain composed entirely of valine, many different single-amino-acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.
    Keywords:  Kaposi Sarcoma Virus; Membrane-associated E3 ubiquitin ligase; deep mutational scanning; immune regulation; membrane protein; protein:protein interactions
  37. EMBO J. 2021 Jun 22. e102509
      The SAGA coactivator complex is essential for eukaryotic transcription and comprises four distinct modules, one of which contains the ubiquitin hydrolase USP22. In yeast, the USP22 ortholog deubiquitylates H2B, resulting in Pol II Ser2 phosphorylation and subsequent transcriptional elongation. In contrast to this H2B-associated role in transcription, we report here that human USP22 contributes to the early stages of stimulus-responsive transcription, where USP22 is required for pre-initiation complex (PIC) stability. Specifically, USP22 maintains long-range enhancer-promoter contacts and controls loading of Mediator tail and general transcription factors (GTFs) onto promoters, with Mediator core recruitment being USP22-independent. In addition, we identify Mediator tail subunits MED16 and MED24 and the Pol II subunit RBP1 as potential non-histone substrates of USP22. Overall, these findings define a role for human SAGA within the earliest steps of transcription.
    Keywords:  SAGA; USP22; epigenetic; pre-initiation complex; transcription
  38. Dis Model Mech. 2021 Jun 23. pii: dmm.048603. [Epub ahead of print]
      Valosin containing protein (VCP) is a hexameric type II AAA ATPase required for several cellular processes including ER-associated degradation, organelle biogenesis, autophagy and membrane fusion. VCP contains three domains: a regulatory N-terminal domain and two ATPase domains (D1 and D2). Mutations in the N-terminal and D1 domains are associated with several degenerative diseases, including Multisystem Proteinopathy (MSP-1) and ALS. However, patients with VCP mutations vary widely in their pathology and clinical penetrance, making it difficult to devise effective treatment strategies. Having a deeper understanding of how each mutation affects VCP function could enhance the prediction of clinical outcomes and design of personalized treatment options. Over-expressing VCP patient mutations in Drosophila has been shown to mimic many pathologies observed in human patients. The power of a genetically tractable model organism coupled with well-established in vivo assays and a relatively short life cycle make Drosophila an attractive system to study VCP disease pathogenesis and novel treatment strategies. Using CRISPR/Cas9, we have generated individual Drosophila knock-in mutants that include nine hereditary VCP disease mutations. We validate that these models display many hallmarks of VCP-mediated degeneration, including progressive decline in mobility, protein aggregate accumulation and defects in lysosomal and mitochondrial function. We also made some novel and unexpected findings, including laminopathies and sex-specific phenotypic differences in several mutants. Taken together, the Drosophila VCP disease models we have generated in this study will be useful for studying the etiology of individual VCP patient mutations and for testing potential genetic and/or pharmacological therapies.
    Keywords:  Drosophila; IBMPFD; Lysosomes; Ter94; VCP; mitochondria
  39. Carcinogenesis. 2021 Jun 19. pii: bgab053. [Epub ahead of print]
      The Homologous to E6AP C-terminus (HECT) domain and RCC1-like domain-containing (HERC) proteins can function as tumour suppressors and as oncogenes, depending on the cancer type. However, the expression patterns of HERCs in colorectal cancer (CRC) cells are unclear. Here, we show that only HERC1 and HERC5 are downregulated in CRC tumours, and we focus our study on revealing HERC5-mediating signalling because the change in downregulation is much more obvious for HERC5 than for HERC1. We demonstrate that HERC5 recruits an adaptor protein, CREB binding protein (CRB), to ubiquitinate C-terminal binding protein 1 (CtBP1) in noncancerous colon cells. The downregulation of HERC5 in CRC cells attenuates the ubiquitination of CtBP1, which then accumulates and assembles into a transcriptional complex with histone deacetylase 1 (HDAC1) and a transcription factor c-MYC. This transcriptional complex binds to the promoters of three proapoptotic genes, Bcl2 associated X (BAX), Bcl2 interacting killer (BIK) and p53upregulated modulator of apoptosis (PUMA), and inhibits their expression, thereby suppressing apoptotic signalling and promoting tumourigenesis. Overexpression of HERC5, downregulation of CtBP1 or blocking of the CtBP1 function with its inhibitors (NSC95397 and 4-methylthio-2-oxobutyric acid [MTOB]) significantly prevents CRC cell proliferation in vitro and tumour growth in vivo. Combining NSC95397 (or MTOB) with chemotherapeutic drugs (oxaliplatin or capecitabine) gives a much stronger inhibition of cell proliferation and tumour growth compared to their single treatments. Collectively, our results reveal that downregulation of HERC5 E3 ligase attenuates the ubiquitination of CtBP1 to inhibit apoptosis. Therefore, CtBP1 may be a promising target in CRC chemotherapy.
    Keywords:  CBP; CtBP1; HERC5 E3 ligase; NSC95397; c-MYC; colorectal cancer
  40. Sci Rep. 2021 Jun 22. 11(1): 13084
      The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.
  41. N Engl J Med. 2021 Jun 24. 384(25): 2406-2417
      BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare.METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast.
    RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7.
    CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).
  42. Mol Cell. 2021 Jun 14. pii: S1097-2765(21)00444-5. [Epub ahead of print]
      OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.
  43. Autophagy. 2021 Jun 22. 1-2
      AMBRA1 (autophagy/beclin 1 regulator 1) is a multifunctional scaffold protein involved in several cellular processes spanning from cell proliferation to apoptosis and to regulation of macroautophagy/autophagy. Our recent publication revealed that Ambra1 has an antitumorigenic role in melanoma, the most aggressive and deadly skin cancer. We have indeed collected data indicating that the increased proliferative and invasive/metastatic features that we observed in ambra1-ablated melanomas are related to a remarkable regulation by Ambra1 on cellular processes which are beyond autophagy. Our study therefore sheds light on intriguing processes affected by Ambra1 which can be exploited as therapeutic targets in AMBRA1 low-expressing melanoma.
    Keywords:  AMBRA1; FAK1; GEMMs; cyclin D1; melanoma; metastasis; proliferation
  44. Elife. 2021 Jun 23. pii: e62718. [Epub ahead of print]10
      The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here, we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate in the cellular model used, DPRs exert additive effects that may contribute to pathology.
    Keywords:  biochemistry; chemical biology; human; neurodegeneration; neuroscience; protein aggregation; quality control
  45. Front Mol Biosci. 2021 ;8 681855
      Cells have evolved a complex molecular network, collectively called the protein homeostasis (proteostasis) network, to produce and maintain proteins in the appropriate conformation, concentration and subcellular localization. Loss of proteostasis leads to a reduction in cell viability, which occurs to some degree during healthy ageing, but is also the root cause of a group of diverse human pathologies. The accumulation of proteins in aberrant conformations and their aggregation into specific beta-rich assemblies are particularly detrimental to cell viability and challenging to the protein homeostasis network. This is especially true for bacteria; it can be argued that the need to adapt to their changing environments and their high protein turnover rates render bacteria particularly vulnerable to the disruption of protein homeostasis in general, as well as protein misfolding and aggregation. Targeting bacterial proteostasis could therefore be an attractive strategy for the development of novel antibacterial therapeutics. This review highlights advances with an antibacterial strategy that is based on deliberately inducing aggregation of target proteins in bacterial cells aiming to induce a lethal collapse of protein homeostasis. The approach exploits the intrinsic aggregation propensity of regions residing in the hydrophobic core regions of the polypeptide sequence of proteins, which are genetically conserved because of their essential role in protein folding and stability. Moreover, the molecules were designed to target multiple proteins, to slow down the build-up of resistance. Although more research is required, results thus far allow the hope that this strategy may one day contribute to the arsenal to combat multidrug-resistant bacterial infections.
    Keywords:  Pept-in; adsorption; advanced oxidation processes; aggregation-prone region; antibacterial peptide; inclusion body; protein aggregation; protein homeostasis
  46. Proc Natl Acad Sci U S A. 2021 Jun 22. pii: e2100690118. [Epub ahead of print]118(25):
      Osteogenesis imperfecta (OI) is a genetic disorder that features wide-ranging defects in both skeletal and nonskeletal tissues. Previously, we and others reported that loss-of-function mutations in FK506 Binding Protein 10 (FKBP10) lead to skeletal deformities in conjunction with joint contractures. However, the pathogenic mechanisms underlying joint dysfunction in OI are poorly understood. In this study, we have generated a mouse model in which Fkbp10 is conditionally deleted in tendons and ligaments. Fkbp10 removal substantially reduced telopeptide lysyl hydroxylation of type I procollagen and collagen cross-linking in tendons. These biochemical alterations resulting from Fkbp10 ablation were associated with a site-specific induction of fibrosis, inflammation, and ectopic chondrogenesis followed by joint deformities in postnatal mice. We found that the ectopic chondrogenesis coincided with enhanced Gli1 expression, indicating dysregulated Hedgehog (Hh) signaling. Importantly, genetic inhibition of the Hh pathway attenuated ectopic chondrogenesis and joint deformities in Fkbp10 mutants. Furthermore, Hh inhibition restored alterations in gait parameters caused by Fkbp10 loss. Taken together, we identified a previously unappreciated role of Fkbp10 in tendons and ligaments and pathogenic mechanisms driving OI joint dysfunction.
    Keywords:  Fkbp10; contracture; ligament; osteogenesis imperfecta; tendon