bims-proteo Biomed News
on Proteostasis
Issue of 2022‒05‒08
38 papers selected by
Eric Chevet

  1. Trends Biochem Sci. 2022 May 01. pii: S0968-0004(22)00088-3. [Epub ahead of print]
      Post-translational modification with ubiquitin is required for cell division, differentiation, and survival in all eukaryotes. As part of an intricate signaling code, ubiquitin is attached to its targets as single molecules or polymeric chains, with the distinct modifications encoding a wide range of outcomes. After early work focused on homotypic ubiquitin chains, such as the K48-linked polymers that drive proteasomal degradation, recent studies noted abundant conjugates that contained ubiquitin molecules modified on two or more sites. Such branched ubiquitin chains are produced in response to specific signals and they exert functions that are critical for cellular and organismal homeostasis. In this review, we will discuss our rapidly evolving understanding of the assembly and function of branched ubiquitin chains.
    Keywords:  DUB; E3 ligase; branched ubiquitin chain; deubiquitylase; heterotypic ubiquitin chain; ubiquitin
  2. Nat Commun. 2022 May 06. 13(1): 2501
      Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates' formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone - BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.
  3. J Mol Biol. 2022 Apr 29. pii: S0022-2836(22)00198-X. [Epub ahead of print] 167618
      The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.
    Keywords:  Mitochondrial Unfolded Protein Response; Molecular Chaperone; Protein misfolding; Proteostasis; Proteotoxic stress; Ribosome Quality Control; Stress Response; TOM complex; Vms1
  4. Autophagy. 2022 May 04. 1-26
      Macroautophagy/autophagy is an essential process for cellular survival and is implicated in many diseases. A critical step in autophagy is the transport of the transcription factor TFEB from the cytosol into the nucleus, through the nuclear pore (NP) by KPNB1/importinβ1. In the C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal lobar degeneration (ALS-FTD), the hexanucleotide (G4C2)RNA expansion (HRE) disrupts the nucleocytoplasmic transport of TFEB, compromising autophagy. Here we show that a molecular chaperone, the SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1), facilitates TFEB transport into the nucleus by chaperoning the NP protein (i.e., nucleoporin) POM121 which recruits KPNB1. In NSC34 cells, HRE reduces TFEB transport by interfering with the association between SIGMAR1 and POM121, resulting in reduced nuclear levels of TFEB, KPNB1, and the autophagy marker LC3-II. Overexpression of SIGMAR1 or POM121, or treatment with the highly selective and potent SIGMAR1 agonist pridopidine, currently in phase 2/3 clinical trials for ALS and Huntington disease, rescues all of these deficits. Our results implicate nucleoporin POM121 not merely as a structural nucleoporin, but also as a chaperone-operated signaling molecule enabling TFEB-mediated autophagy. Our data suggest the use of SIGMAR1 agonists, such as pridopidine, for therapeutic development of diseases in which autophagy is impaired.Abbreviations: ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementiaC9ALS-FTD, C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal dementiaCS, citrate synthaseER, endoplasmic reticulumGSS, glutathione synthetaseHRE, hexanucleotide repeat expansionHSPA5/BiP, heat shock protein 5LAMP1, lysosomal-associated membrane protein 1MAM, mitochondria-associated endoplasmic reticulum membraneMAP1LC3/LC3, microtubule-associated protein 1 light chain 3NP, nuclear poreNSC34, mouse motor neuron-like hybrid cell lineNUPs, nucleoporinsPOM121, nuclear pore membrane protein 121SIGMAR1/Sigma-1R, sigma non-opioid intracellular receptor 1TFEB, transcription factor EBTMEM97/Sigma-2R, transmembrane protein 97.
    Keywords:  ALS/FTD; KPNB1/importinβ1; SIGMAR1; TFEB; c9orf72; chaperone; nucleocytoplasmic transport; nucleoporin POM121; pridopidine; sigma-1 receptor
  5. J Biol Chem. 2022 Apr 29. pii: S0021-9258(22)00437-9. [Epub ahead of print] 101997
      Inositol-requiring enzyme 1 (IRE1) is an evolutionarily conserved sensor of endoplasmic reticulum (ER) stress and mediates a key branch of the unfolded protein response (UPR) in eukaryotic cells. It is an ER-resident transmembrane protein that possesses Ser/Thr protein kinase and endoribonuclease (RNase) activities in its cytoplasmic region. IRE1 is activated through dimerization/oligomerization and autophosphorylation at multiple sites, acting through its RNase activity to restore the functional capacity of the ER. However, it remains poorly defined in vivo how the autophosphorylation events of endogenous IRE1 govern its dynamic activation and functional output. Here we generated a mouse model harboring a S724A knock-in mutation (Ern1S724A/S724A) and investigated the importance of phosphorylation at Ser724 located within the kinase activation loop of murine IRE1α. We found that in mouse embryonic fibroblast (MEF) cells as well as in primary hepatocytes, S724A mutation resulted in markedly reduced rate of IRE1α autophosphorylation in parallel with blunted activation of its RNase activity to catalyze Xbp1 mRNA splicing. Furthermore, ablation of IRE1α phosphorylation at Ser724 exacerbated ER stress-induced hepatic steatosis in Tunicamycin-treated Ern1S724A/S724A mice. This was accompanied by significantly decreased production of XBP1s protein but increased CHOP protein levels in the liver, along with suppressed expression of key metabolic regulators of fatty acid β-oxidation and lipid secretion. These results demonstrate a critical role of phosphorylation at Ser724 of IRE1α in dynamically controlling its kinase activity, and thus its autophosphorylation state, which is coupled to the activation of its RNase activity in counteracting hepatic steatosis under ER stress conditions.
    Keywords:  ER stress; Hepatic steatosis; IRE1α; RIDD; Xbp1; autophosphorylation
  6. Trends Biochem Sci. 2022 Apr 29. pii: S0968-0004(22)00092-5. [Epub ahead of print]
      The signal recognition particle (SRP) cotranslationally targets a large and diverse portion of the nascent proteome to the endoplasmic reticulum (ER). A recent study by Jomaa et al. reveals an unexpected function for the ribosome-bound nascent chain-associated complex (NAC) in sensing ER-targeting signals and recruiting SRP to the appropriate ribosomes for high-fidelity targeting.
  7. Biochem Biophys Res Commun. 2022 Apr 25. pii: S0006-291X(22)00636-2. [Epub ahead of print]612 44-49
      Oligomannose-type glycans on glycoproteins play an important role in the endoplasmic reticulum (ER)-protein quality control. Mannose trimming of the glycans triggers the ER-associated protein degradation pathway. In mammals, ER mannosyl-oligosaccharide 1,2-α-mannosidase 1 and three ER degradation -enhancing α-mannosidase-like proteins (EDEMs) are responsible for mannose trimming. However, the exact role of EDEMs as α-mannosidases in ERAD remains unclear. Here, we performed the biochemical characterization of EDEM3 using synthetic oligomannose-type glycan substrates. In vitro assays revealed that EDEM3 can convert an asparagine-linked M9 glycan to M8 and M7 glycans in contrast to glycine-linked M9 glycan, and the activity is enhanced in the presence of ERp46, a known partner protein of EDEM3. Our study provides novel insights into the enzymatic properties of EDEM3 and the use of artificial glycan substrates as tools to study ERAD mechanisms.
    Keywords:  EDEM3; ERAD; Glycoprotein; Oligomannose-type glycans; Protein quality control
  8. Mol Neurobiol. 2022 Apr 30.
      Charcot-Marie-Tooth disease type 1A (CMT1A), caused by duplication of the peripheral myelin protein 22 (PMP22) gene, and CMT1B, caused by mutations in myelin protein zero (MPZ) gene, are the two most common forms of demyelinating CMT (CMT1), and no treatments are available for either. Prior studies of the MpzSer63del mouse model of CMT1B have demonstrated that protein misfolding, endoplasmic reticulum (ER) retention and activation of the unfolded protein response (UPR) contributed to the neuropathy. Heterozygous patients with an arginine to cysteine mutation in MPZ (MPZR98C) develop a severe infantile form of CMT1B which is modelled by MpzR98C/ + mice that also show ER stress and an activated UPR. C3-PMP22 mice are considered to effectively model CMT1A. Altered proteostasis, ER stress and activation of the UPR have been demonstrated in mice carrying Pmp22 mutations. To determine whether enabling the ER stress/UPR and readjusting protein homeostasis would effectively treat these models of CMT1B and CMT1A, we administered Sephin1/IFB-088/icerguestat, a UPR modulator which showed efficacy in the MpzS63del model of CMT1B, to heterozygous MpzR98C and C3-PMP22 mice. Mice were analysed by behavioural, neurophysiological, morphological and biochemical measures. Both MpzR98C/ + and C3-PMP22 mice improved in motor function and neurophysiology. Myelination, as demonstrated by g-ratios and myelin thickness, improved in CMT1B and CMT1A mice and markers of UPR activation returned towards wild-type values. Taken together, our results demonstrate the capability of IFB-088 to treat a second mouse model of CMT1B and a mouse model of CMT1A, the most common form of CMT. Given the recent benefits of IFB-088 treatment in amyotrophic lateral sclerosis and multiple sclerosis animal models, these data demonstrate its potential in managing UPR and ER stress for multiple mutations in CMT1 as well as in other neurodegenerative diseases. (Left panel) the accumulation of overexpressed PMP22 or misfolded mutant P0 in the Schwann cell endoplasmic reticulum (ER) leads to overwhelming of the degradative capacity, activation of ER-stress mechanisms, and myelination impairment. (Right panel) by prolonging eIF2α phosphorylation, IFB-088 reduces the amount of newly synthesized proteins entering the ER, allowing the protein quality control systems to better cope with the unfolded/misfolded protein and allowing myelination to progress.
    Keywords:  Charcot-Marie-Tooth; IFB-088/Sephin1/icerguestat; Neuropathy; Proteostasis; UPR
  9. Cell Rep. 2022 05 03. pii: S2211-1247(22)00532-0. [Epub ahead of print]39(5): 110768
      Glycosylphosphatidylinositol-anchored proteins (GPI-APs) exit the endoplasmic reticulum (ER) through a specialized export pathway in the yeast Saccharomyces cerevisiae. We have recently shown that a very-long acyl chain (C26) ceramide present in the ER membrane drives clustering and sorting of GPI-APs into selective ER exit sites (ERES). Now, we show that this lipid-based ER sorting also involves the C26 ceramide as a lipid moiety of GPI-APs, which is incorporated into the GPI anchor through a lipid-remodeling process after protein attachment in the ER. Moreover, we also show that a GPI-AP with a C26 ceramide moiety is monitored by the GPI-glycan remodelase Ted1, which, in turn, is required for receptor-mediated export of GPI-APs. Therefore, our study reveals a quality-control system that ensures lipid-based sorting of GPI-APs into selective ERESs for differential ER export, highlighting the physiological need for this specific export pathway.
    Keywords:  CP: Cell biology; CP: Molecular biology; GPI-anchored protein; ceramide remodeling; endoplasmic reticulum; glycan remodeling; protein sorting; quality control; yeast Saccharomyces cerevisiae
  10. Autophagy. 2022 May 04. 1-20
      Mutations in DNAJC5/CSPα are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. Here we report two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AFSM: autofluorescent storage materials; ANCL: adult neuronal ceroid lipofuscinosis; Baf. A1: bafilomycin A1; CLN: ceroid lipofuscinosis neuronal; CLU: clusterin; CS: cysteine string domain of DNAJC5/CSPα; CUPS: compartment for unconventional protein secretion; DN: dominant negative; DNAJC5/CSPα: DnaJ heat shock protein family (Hsp40) member C5; eMI: endosomal microautophagy; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INCL: infant neuronal ceroid lipofuscinosis; JNCL: juvenile neuronal ceroid lipofuscinosis; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAPTM4B: lysosomal protein transmembrane 4 beta; LN: linker domain of DNAJC5/CSPα; MAPS: misfolding-associated protein secretion; mCh/Ch: mCherry; mCi/Ci: mCitrine; MTOR: mechanistic target of rapamycin kinase; NCL: neuronal ceroid lipofuscinosis; PPT1: palmitoyl-protein thioesterase 1; PQC: protein quality control; SBP: streptavidin binding protein; SGT: small glutamine-rich tetratricopeptide repeat; shRNA: short hairpin RNA; SLC3A2/CD98hc: solute carrier family 3 member 2; SNCA/α-synuclein: synuclein alpha; TMED10: transmembrane p24 trafficking protein 10; UV: ultraviolet; VPS4: vacuolar protein sorting 4 homolog; WT: wild type.
    Keywords:  CLN4; DNAJC5/CSPα; ESCRT; cysteine string protein α; lysobody; lysosome/endolysosome; microautophagy/eMI; misfolding-associated protein secretion/MAPS; neuronal ceroid lipofuscinosis/NCL; protein quality control/PQC; unconventional protein secretion/UPS
  11. RNA Biol. 2022 Jan;19(1): 609-621
      Cells of metazoans respond to internal and external stressors by activating stress response pathways that aim for re-establishing cellular homoeostasis or, if this cannot be achieved, triggering programmed cell death. Problems during translation, arising from defective mRNAs, tRNAs, ribosomes or protein misfolding, can activate stress response pathways as well as mRNA surveillance and ribosome quality control programs. Recently, ribosome collisions have emerged as a central signal for translational stress and shown to elicit different stress responses. Here, we review our current knowledge about the intricate mutual connections between ribosome collisions, stress response pathways and mRNA surveillance. A central factor connecting the sensing of collided ribosomes with degradation of the nascent polypeptides, dissociation of the stalled ribosomes and degradation of the mRNA by no-go or non-stop decay is the E3-ligase ZNF598. We tested whether ZNF598 also plays a role in nonsense-mediated mRNA decay (NMD) but found that it is dispensable for this translation termination-associated mRNA surveillance pathway, which in combination with other recent data argues against stable ribosome stalling at termination codons being the NMD-triggering signal.
    Keywords:  ISR; NGD; NMD; NSD; RQC; Stress response; UPR; ZNF598; mRNA surveillance; quality control; ribosome; ribosome collisions; ribotoxic stress; translation
  12. Autophagy. 2022 May 06. 1-15
      Macroautophagy/autophagy, an evolutionarily conserved degradation system, serves to clear intracellular components through the lysosomal pathway. Mounting evidence has revealed cytoprotective roles of autophagy; however, the intracellular causes of overactivated autophagy, which has cytotoxic effects, remain elusive. Here we show that sustained proteotoxic stress induced by loss of the RING and Kelch repeat-containing protein C53A5.6/RIKE-1 induces sequestration of LET-363/MTOR complex and overactivation of autophagy, and consequently impairs epithelial integrity in C. elegans. In C53A5.6/RIKE-1-deficient animals, blocking autophagosome formation effectively prevents excessive endosomal degradation, mitigates mislocalization of intestinal membrane components and restores intestinal lumen morphology. However, autophagy inhibition does not affect LET-363/MTOR aggregation in animals with compromised C53A5.6/RIKE-1 function. Improving proteostasis capacity by reducing DAF-2 insulin/IGF1 signaling markedly relieves the aggregation of LET-363/MTOR and alleviates autophagy overactivation, which in turn reverses derailed endosomal trafficking and rescues epithelial morphogenesis defects in C53A5.6/RIKE-1-deficient animals. Hence, our studies reveal that C53A5.6/RIKE-1-mediated proteostasis is critical for maintaining the basal level of autophagy and epithelial integrity.Abbreviations: ACT-5: actin 5; ACTB: actin beta; ALs: autolysosomes; APs: autophagosomes; AJM-1: apical junction molecule; ATG: autophagy related; C. elegans: Caenorhabditis elegans; CPL-1: cathepsin L family; DAF: abnormal dauer formation; DLG-1: Drosophila discs large homolog; ERM-1: ezrin/radixin/moesin; EPG: ectopic P granule; GFP: freen fluorescent protein; HLH-30: helix loop helix; HSP: heat shock protein; LAAT-1: lysosome associated amino acid transporter; LET: lethal; LGG-1: LC3, GABARAP and GATE-16 family; LMP-1: LAMP (lysosome-associated membrane protein) homolog; MTOR: mechanistic target of rapamycin kinase; NUC-1: abnormal nuclease; PEPT-1/OPT-2: Peptide transporter family; PGP-1: P-glycoprotein related; RAB: RAB family; RIKE-1: RING and Kelch repeat-containing protein; SLCF-1: solute carrier family; SQST-1: sequestosome related; SPTL-1: serine palmitoyl transferase family.
    Keywords:  Autophagy; C. elegans; LET-363/MTOR; endosomal degradation; epithelial morphogenesis; proteostasis
  13. Hepatology. 2022 May 06.
      The endoplasmic reticulum (ER) is an intracellular organelle that fosters the correct folding of linear polypeptides and proteins, a process tightly governed by the ER-resident enzymes and chaperones. Failure to shape the proper 3-dimensional architecture of proteins culminates in the accumulation of misfolded or unfolded proteins within the ER, disturbs ER homeostasis, and leads to canonically defined ER stress. Recent studies have elucidated that cellular perturbations, such as lipotoxicity, can also lead to ER stress. In response to ER stress, the unfolded protein response (UPR) is activated to reestablish ER homeostasis ("adaptive UPR") or conversely, to provoke cell death when ER stress is overwhelmed and sustained ("maladaptive UPR"). It is well documented that ER stress contributes to the onset and progression of multiple hepatic pathologies including non-alcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), viral hepatitis, liver ischemia, drug toxicity, and liver cancers. Here, we review key studies dealing with the emerging role of ER stress and the UPR in the pathophysiology of liver diseases from cellular, murine, and human models. Specifically, we will summarize current available knowledge on pharmacological and non-pharmacological interventions that may be employed to target maladaptive UPR for the treatment of non-malignant liver diseases.
    Keywords:  ER stress; Liver cancer; Liver disease; Liver toxicity; Liver viral infections; UPR
  14. PLoS Pathog. 2022 May 02. 18(5): e1009717
      The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.
  15. Biol Futur. 2022 May 02.
      Lysosomal-dependent self-degradative (autophagic) mechanisms are essential for the maintenance of normal homeostasis in all eukaryotic cells. Several types of such self-degradative and recycling pathways have been identified, based on how the cellular self material can incorporate into the lysosomal lumen. Ubiquitination, a well-known and frequently occurred posttranslational modification has essential role in all cell biological processes, thus in autophagy too. The second most common type of polyubiquitin chain is the K63-linked polyubiquitin, which strongly connects to some self-degradative mechanisms in the cells. In this review, we discuss the role of this type of polyubiquitin pattern in numerous autophagic processes.
    Keywords:  Lysine 63 linkage specific ubiquitin; Lysosome; Vesicular trafficking
  16. EMBO J. 2022 May 02. e109460
      PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observed that phospho-ubiquitin can bind to two distinct sites on parkin, a high-affinity site on RING1 that controls parkin localization and a low-affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.
    Keywords:  Parkinson's disease; autophagy; mitophagy; open-loop control; ubiquitin
  17. Front Plant Sci. 2022 ;13 873688
      Endoplasmic Reticulum-Associated Degradation (ERAD) is one of the major processes in maintaining protein homeostasis. Class I α-mannosidases MNS4 and MNS5 are involved in the degradation of misfolded variants of the heavily glycosylated proteins, playing an important role for glycan-dependent ERAD in planta. MNS4 and MNS5 reportedly have functional redundancy, meaning that only the loss of both MNS4 and MNS5 shows phenotypes. However, MNS4 is a membrane-associated protein while MNS5 is a soluble protein, and both can localize to the endoplasmic reticulum (ER). Furthermore, MNS4 and MNS5 differentially demannosylate the glycoprotein substrates. Importantly, we found that their gene expression patterns are complemented rather than overlapped. This raises the question of whether they indeed work redundantly, warranting a further investigation. Here, we conducted an exhaustive genetic screen for a suppressor of the bri1-5, a brassinosteroid (BR) receptor mutant with its receptor downregulated by ERAD, and isolated sbi3, a suppressor of bri1-5 mutant named after sbi1 (suppressor of bri1). After genetic mapping together with whole-genome re-sequencing, we identified a point mutation G343E in AT1G27520 (MNS5) in sbi3. Genetic complementation experiments confirmed that sbi3 was a loss-of-function allele of MNS5. In addition, sbi3 suppressed the dwarf phenotype of bri1-235 in the proteasome-independent ERAD pathway and bri1-9 in the proteasome-dependent ERAD pathway. Importantly, sbi3 could only affect BRI1/bri1 with kinase activities such that it restored BR-sensitivities of bri1-5, bri1-9, and bri1-235 but not null bri1. Furthermore, sbi3 was less tolerant to tunicamycin and salt than the wild-type plants. Thus, our study uncovers a non-redundant function of MNS5 in the regulation of ERAD as well as plant growth and ER stress response, highlighting a need of the traditional forward genetic approach to complement the T-DNA or CRISPR-Cas9 systems on gene functional study.
    Keywords:  BRI1; ERAD; MNS4; MNS5; SBI3
  18. Front Aging Neurosci. 2022 ;14 854380
      Insoluble protein deposits are hallmarks of neurodegenerative disorders and common forms of dementia. The aberrant aggregation of misfolded proteins involves a complex cascade of events that occur over time, from the cellular to the clinical phase of neurodegeneration. Declining neuronal health through increased cell stress and loss of protein homeostasis (proteostasis) functions correlate with the accumulation of aggregates. On the cellular level, increasing evidence supports that misfolded proteins may undergo liquid-liquid phase separation (LLPS), which is emerging as an important process to drive protein aggregation. Studying the reverse process of aggregate disassembly and degradation has only recently gained momentum, following reports of enzymes with distinct aggregate-disassembly activities. In this review, we will discuss how the ubiquitin-proteasome system and disaggregation machineries such as VCP/p97 and HSP70 system may disassemble and/or degrade protein aggregates. In addition to their canonically associated functions, these enzymes appear to share a common feature: reversibly assembling into liquid droplets in an LLPS-driven manner. We review the role of LLPS in enhancing the disassembly of aggregates through locally increasing the concentration of these enzymes and their co-proteins together within droplet structures. We propose that such activity may be achieved through the concerted actions of disaggregase machineries, the ubiquitin-proteasome system and their co-proteins, all of which are condensed within transient aggregate-associated droplets (TAADs), ultimately resulting in aggregate clearance. We further speculate that sustained engagement of these enzymatic activities within TAADs will be detrimental to normal cellular functions, where these activities are required. The possibility of facilitating endogenous disaggregation and degradation activities within TAADs potentially represents a novel target for therapeutic intervention to restore protein homeostasis at the early stages of neurodegeneration.
    Keywords:  disaggregase machinery; liquid droplet; liquid-liquid phase separation; neurodegenaration; protein aggregation; protein misfolding; ubiquitin proteasome pathway
  19. J Cell Biol. 2022 Jul 04. pii: e202111100. [Epub ahead of print]221(7):
      The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR's sensor domain and by front-to-front interfaces between PKR's kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.
  20. Exp Mol Med. 2022 May 05.
      Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor β (IRβ). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRβ in hepatocytes.
  21. J Proteomics. 2022 Apr 27. pii: S1874-3919(22)00116-6. [Epub ahead of print]262 104592
      The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14 (RPN11), USP14 and UCH37 (UCHL5). However, the functional roles and specificities of these proteasomal DUBs remain elusive. To reveal the specificities of proteasome associated DUBs, we used SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. We observed distinct effects on the global ubiquitinome upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggested less functional redundancy than previously anticipated. We also investigated whether the small molecule inhibitor b-AP15 has the potential to specifically target USP14 and UCH37 by comparing treatment of wild-type versus USP14/UCH37 double-knockout cells with this drug. Strikingly, broad and severe off-target effects were observed, questioning the alleged specificity of this inhibitor. In conclusion, this work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.SIGNIFICANCE: Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive.
    MATERIALS & METHODS: We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37.
    RESULTS: We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor.
    CONCLUSIONS: This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.
    Keywords:  Deubiquitinase (DUB); Parallel reaction monitoring; Proteasome; Quantitative mass spectrometry; SILAC; UCH37/UCHL5; USP14; Ubiquitin; Ubiquitinome; Ubiquitinomics
  22. Nucleic Acids Res. 2022 Apr 30. pii: gkac304. [Epub ahead of print]
      Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.
  23. Nat Cell Biol. 2022 May 02.
      Innate DNA sensing via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) mechanism surveys microbial invasion and cellular damage and thus participates in various human infectious diseases, autoimmune diseases and cancers. However, how DNA sensing rapidly and adaptively shapes cellular physiology is incompletely known. Here we identify the STING-PKR-like endoplasmic reticulum kinase (PERK)-eIF2α pathway, a previously unknown cGAS-STING mechanism, enabling an innate immunity control of cap-dependent messenger RNA translation. Upon cGAMP binding, STING at the ER binds and directly activates the ER-located kinase PERK via their intracellular domains, which precedes TBK1-IRF3 activation and is irrelevant to the unfolded protein response. The activated PERK phosphorylates eIF2α, forming an inflammatory- and survival-preferred translation program. Notably, this STING-PERK-eIF2α pathway is evolutionarily primitive and physiologically critical to cellular senescence and organ fibrosis. Pharmacologically or genetically targeting this non-canonical cGAS-STING pathway attenuated lung and kidney fibrosis. Collectively, the findings identify an alternative innate immune pathway and its critical role in organ fibrosis, report an innate immunity-directed translation program and suggest the therapeutic potential for targeting the STING-PERK pathway in treating fibrotic diseases.
  24. J Cell Biol. 2022 Jun 06. pii: e202105112. [Epub ahead of print]221(6):
      Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors associated with non-canonical ATG16L1 recruitment and CASM induction remain unknown. Here, using pharmacological inhibitors, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM and indicate that SopF evolved in part to evade non-canonical autophagy.
  25. Life Sci Alliance. 2022 Sep;pii: e202101346. [Epub ahead of print]5(9):
      Rab40c is a SOCS box-containing protein which binds Cullin5 to form a ubiquitin E3 ligase complex (Rab40c/CRL5) to regulate protein ubiquitylation. However, the exact functions of Rab40c remain to be determined, and what proteins are the targets of Rab40c-Cullin5-mediated ubiquitylation in mammalian cells are unknown. Here we showed that in migrating MDA-MB-231 cells Rab40c regulates focal adhesion's number, size, and distribution. Mechanistically, we found that Rab40c binds the protein phosphatase 6 (PP6) complex and ubiquitylates one of its subunits, ankyrin repeat domain 28 (ANKRD28), thus leading to its lysosomal degradation. Furthermore, we identified that phosphorylation of FAK and MOB1 is decreased in Rab40c knock-out cells, which may contribute to focal adhesion site regulation by Rab40c. Thus, we propose a model where Rab40c/CRL5 regulates ANKRD28 ubiquitylation and degradation, leading to a decrease in PP6 activity, which ultimately affects FAK and Hippo pathway signaling to alter focal adhesion dynamics.
  26. J Mater Chem B. 2022 May 04.
      Ubiquitination is a prevalent post-translational modification that controls a multitude of important biological processes. Due to the low abundance of ubiquitinated proteins, highly efficient separation and enrichment approaches are required for ubiquitinome analysis. In this work, we disclose the region-specific interactions between the hydrophobic patch of ubiquitin and polydopamine. Taking advantage of this inherent binding property, we have constructed surface-imprinted magnetic nanoparticles (NPs) for ubiquitin by sequential dopamine polymerization and surface PEGylation. The obtained molecularly imprinted polymer (MIP) NPs showed a binding constant of 2.6 × 106 L mol-1 for the template ubiquitin. The bound ubiquitin could be quantitatively released by heating to 70 °C at pH 2.0 or 90 °C at neutral (pH 7.0) conditions. The MIP NPs exhibited nano receptor-like property which not only effectively blocked the formation of branched ubiquitin chains but also selectively separated ubiquitin from the bacterial cell lysates. By incubating the MIP NPs with the lysates of 293T cells, totally 529 ubiquitinated proteins were captured, among which 287 proteins were not identified by the anti-ubiquitin monoclonal antibodies (mAbs). With the distinct merits of low cost and high stability, the as-prepared MIP NPs may be utilized either separately or as an important complement to the mAbs for the purification and enrichment of ubiquitin and ubiquitinated proteins from complex biological samples. Furthermore, due to the flexibility in modification of the binding sites during or after the imprinting reactions, the results of this work also paved the way for generation of artificial receptors for branched ubiquitin chains and polyubiquitinated proteins with higher avidity and specificity.
  27. J Am Chem Soc. 2022 May 06.
      The tumor suppressor protein fragile histidine triad (Fhit) is known to be associated with genomic instability and apoptosis. The tumor-suppressive function of Fhit depends on the interaction with the alarmone diadenosine triphosphate (Ap3A), a noncanonical nucleotide whose concentration increases upon cellular stress. How the Fhit-Ap3A complex exerts its signaling function is unknown. Here, guided by a chemical proteomics approach employing a synthetic stable Fhit-Ap3A complex, we found that the Fhit-Ap3A complex, but not Fhit or Ap3A alone, impedes translation. Our findings provide a mechanistic model in which Fhit translocates from the nucleolus into the cytosol upon stress to form an Fhit-Ap3A complex. The Fhit-Ap3A complex impedes translation both in vitro and in vivo, resulting in reduced cell viability. Overall, our findings provide a mechanistic model by which the tumor suppressor Fhit collaborates with the alarmone Ap3A to regulate cellular proliferation.
  28. Autophagy Rep. 2022 ;1(1): 79-82
      In the final critical step for autophagic degradation, lysosomes fuse with autophagosomes to form autolysosomes. Although recent research has suggested that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are important for lysosome-autophagosome fusion, neither the architecture of the prefusion state nor the regulatory mechanisms have been identified. In our study, using structured illumination microscopy, we observed that lysosomes formed clusters around individual autophagosomes, thereby setting the stage for membrane fusion. Moreover, VAMP8 (vesicle-associated membrane protein 8) assists in forming the prefusion state of these clusters. We also found that VAMP8 phosphorylation reduces spontaneous lysosome-autophagosome fusion, whereas its dephosphorylation promotes fusion events between lysosomes and autophagosomes in both normal and autophagy-induced conditions. Our data thus suggest a key role of VAMP8 phosphorylation in the regulation of lysosome-autophagosome fusion.
    Keywords:  Autophagy; VAMP8; fusion; lysosomes; phosphorylation
  29. J Cell Biol. 2022 Jul 04. pii: e202202060. [Epub ahead of print]221(7):
      The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.
  30. Oncoimmunology. 2022 ;11(1): 2068488
      Interferon gamma (IFNG/IFNγ)-induced adaptive immune resistance remains a challenge for tumor therapy. We observed that the chaperone heat shock protein 90 (HSP90) stabilizes the transcription factor signal transducer and activator of transcription 1 (STAT1), resulting in IFNγ-induced expression of immunosuppressive indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1/CD274). Pharmacological inhibition of HSP90 enhances the efficacy of programmed cell death 1 (PDCD1/PD-1) targeting immunotherapy in suitable mouse models without any toxicity.
    Keywords:  Adaptive immune resistance; immune checkpoint; molecular chaperone; pancreatic cancer; protein degradation
  31. Trends Biochem Sci. 2022 Apr 27. pii: S0968-0004(22)00086-X. [Epub ahead of print]
      In recent years, a surprisingly complex picture emerged about endoplasmic reticulum (ER)/Golgi-independent secretory pathways, and several routes have been discovered that differ with regard to their molecular mechanisms and machineries. Fibroblast growth factor 2 (FGF2) is secreted by a pathway of unconventional protein secretion (UPS) that is based on direct self-translocation across the plasma membrane. Building on previous research, a component of this process has been identified to be glypican-1 (GPC1), a GPI-anchored heparan sulfate proteoglycan located on cell surfaces. These findings not only shed light on the molecular mechanism underlying this process but also reveal an intimate relationship between FGF2 and GPC1 that might be of critical relevance for the prominent roles they both have in tumor progression and metastasis.
    Keywords:  fibroblast growth factor 2 (FGF2); glypican-1 (GPC1); membrane nanodomains; protein translocation; unconventional protein secretion (UPS)
  32. Nucleic Acids Res. 2022 May 02. pii: gkac257. [Epub ahead of print]
      The -1 programmed ribosomal frameshifting (-1 PRF) has been explored as a gene regulatory circuit for synthetic biology applications. The -1 PRF usually uses an RNA pseudoknot structure as the frameshifting stimulator. Finding a ligand-responsive pseudoknot with efficient -1 PRF activity is time consuming and is becoming a bottleneck for its development. Inserting a guanine to guanine (GG)-mismatch pair in the 5'-stem of a small frameshifting pseudoknot could attenuate -1 PRF activity by reducing stem stability. Thus, a ligand-responsive frameshifting pseudoknot can be built using GG-mismatch-targeting small molecules to restore stem stability. Here, a pseudoknot requiring stem-loop tertiary interactions for potent frameshifting activity was used as the engineering template. This considerably amplified the effect of mismatch destabilization, and led to creation of a mammalian -1 PRF riboswitch module capable of mediating premature translation termination as a synthetic regulatory mode. Application of the synthetic circuit allowed ligand-dependent ATF6N mimic formation for the activation of protein folding-related genes involved in the unfolded protein response without an ER-stress inducing agent. With the availability of mismatch-targeting molecules, the tailored module thus paves the way for various mismatch plug-ins to streamline highly efficient orthogonal ligand-dependent -1 PRF stimulator development in the synthetic biology toolbox.
  33. Nat Commun. 2022 May 05. 13(1): 2491
      Small RNAs include tRNA, snRNA, micro-RNA, tRNA fragments and others that constitute > 90% of RNA copy numbers in a human cell and perform many essential functions. Popular small RNA-seq strategies limit the insights into coordinated small RNA response to cellular stress. Small RNA-seq also lacks multiplexing capabilities. Here, we report a multiplex small RNA-seq library preparation method (MSR-seq) to investigate cellular small RNA and mRNA response to heat shock, hydrogen peroxide, and arsenite stress. Comparing stress-induced changes of total cellular RNA and polysome-associated RNA, we identify a coordinated tRNA response that involves polysome-specific tRNA abundance and synergistic N3-methylcytosine (m3C) tRNA modification. Combining tRNA and mRNA response to stress we reveal a mechanism of stress-induced down-regulation in translational elongation. We also find that native tRNA molecules lacking several modifications are biased reservoirs for the biogenesis of tRNA fragments. Our results demonstrate the importance of simultaneous investigation of small RNAs and their modifications in response to varying biological conditions.
  34. Cell Rep. 2022 05 03. pii: S2211-1247(22)00531-9. [Epub ahead of print]39(5): 110767
      Regulated loading of eIF3-bound 40S ribosomes on capped mRNA is generally dependent upon the translation initiation factor eIF4E; however, mRNA translation often proceeds during physiological stress, such as virus infection, when eIF4E availability and activity are limiting. It remains poorly understood how translation of virus and host mRNAs are regulated during infection stress. While initially sensitive to mTOR inhibition, which limits eIF4E-dependent translation, we show that protein synthesis in human cytomegalovirus (HCMV)-infected cells unexpectedly becomes progressively reliant upon eIF3d. Targeting eIF3d selectively inhibits HCMV replication, reduces polyribosome abundance, and interferes with expression of essential virus genes and a host gene expression signature indicative of chronic ER stress that fosters HCMV reproduction. This reveals a strategy whereby cellular eIF3d-dependent protein production is hijacked to exploit virus-induced ER stress. Moreover, it establishes how switching between eIF4E and eIF3d-responsive cap-dependent translation can differentially tune virus and host gene expression in infected cells.
    Keywords:  CP: Microbiology; CP: Molecular biology; eIF3d; human cytomegalovirus; infection biology; physiological stress; regulation of translation
  35. EMBO Mol Med. 2022 May 02. e14121
      The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase-8 as a pro-survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ-specific manner. In particular, we find that deletion of Caspase-8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase-8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut-vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function.
    Keywords:  caspase-8; chronic intestinal inflammation; endothelium; necroptosis; vascular homeostasis
  36. Cancer Lett. 2022 Apr 29. pii: S0304-3835(22)00200-2. [Epub ahead of print] 215716
      Proteolysis-targeting chimeras (PROTACs) are small molecules that specifically link E3 ubiquitin ligases to proteins of interest to mediate targeted ubiquitination and degradation. PROTACs are advantageous since they can target undruggable proteins with multiple domains, particularly those with smooth surfaces that lack a common binding domain for small-molecule inhibitors (SMIs). This review provides an overview of PROTAC technology and third-generation PROTAC development. We focused on designing and executing the most recent clinical trials involving PROTACs in cancer therapy. Additionally, we summarized novel findings regarding the mechanisms and signaling pathways involved in cancer development, such as the scaffolding function of certain proteins ignored by traditional SMIs and several recognized oncoproteins that participate in novel signaling pathways. We also discussed strategies for enhancing PROTAC antitumor activity and specificity.
    Keywords:  Carcinoma; E3 ligase; PROTAC; Ubiquitin-proteasome system; Undruggable protein
  37. STAR Protoc. 2022 Jun 17. 3(2): 101339
      Valosin-containing protein (VCP, also known as p97/Cdc48) comprises six identical 97 kDa VCP protomers and functions as a master regulator of cellular homeostasis. VCP dodecamer in an apo nucleotide status was recently reported, providing a new framework for studying VCP's diverse biological functions. Here, we present a detailed protocol for purifying and cryo-EM structurally characterizing VCP dodecamers from both bacterial and mammalian cells. This protocol can also be adapted to yeast Cdc48. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).
    Keywords:  Cryo-EM; Molecular Biology; Protein Biochemistry; Protein expression and purification; Structural Biology
  38. Cell Rep Methods. 2022 Apr 25. 2(4): 100198
      We introduce Metis, a new plugin for the Perseus software aimed at analyzing quantitative multi-omics data based on metabolic pathways. Data from different omics types are connected through reactions of a genome-scale metabolic-pathway reconstruction. Metabolite concentrations connect through the reactants, while transcript, protein, and protein post-translational modification (PTM) data are associated through the enzymes catalyzing the reactions. Supported experimental designs include static comparative studies and time-series data. As an example for the latter, we combine circadian mouse liver multi-omics data and study the contribution of cycles of phosphoproteome and metabolome to enzyme activity regulation. Our analysis resulted in 52 pairs of cycling phosphosites and metabolites connected through a reaction. The time lags between phosphorylation and metabolite peak show non-uniform behavior, indicating a major contribution of phosphorylation in the modulation of enzymatic activity.
    Keywords:  Metis; Perseus; circadian rhythms; enzyme activity regulation; metabolic networks; metabolomics; multi-omics; phosphoproteomics; proteomics; transcriptomics