bims-proteo Biomed News
on Proteostasis
Issue of 2021‒10‒31
28 papers selected by
Eric Chevet

  1. Autophagy. 2021 Oct 28. 1-11
      The pathogenesis of pancreatitis has been linked to disruption of organelle homeostasis including macroautophagy/autophagy dysfunction and endoplasmic reticulum (ER) stress. However, the direct impact of aberrant organelle function on pancreatitis initiation and progression is largely unknown. Recently an ER membrane protein, VMP1 (vacuole membrane protein 1), has been reported to play a crucial role in autophagosome formation. Notably, we found that VMP1 is downregulated in both human chronic pancreatitis (CP) and experimental mouse acute pancreatitis (AP). Pancreatic acinar cell-specific vmp1 deletion promotes inflammation, acinar-to-ductal metaplasia, and fibrosis in mice, sharing histological similarities with human CP. Mechanistically, loss of pancreatic VMP1 leads to defective autophagic degradation and ER stress as well as activation of the NFE2L2/Nrf2 pathway. Genetic ablation of NFE2L2 attenuated pancreatitis in VMP1-deficient mice. Our data highlight the importance of VMP1 in modulating an integrated organelle stress response and its functional role in maintaining pancreas homeostasis in the context of CP.Abbreviations: AMY: amylase; ADM: acinar-to-ductal metaplasia; AP: acute pancreatitis; CASP3: caspase 3; CP: chronic pancreatitis; DDIT3/CHOP: DNA damage inducible transcript 3; DKO, double knockout; ER: endoplasmic reticulum; GCLC: glutamate-cysteine ligase catalytic subunit; GCLM: glutamate-cysteine ligase modifier subunit; HSPA5/BIP: heat shock protein family A (Hsp70) member 5; KO: knockout; KRT19/CK19: keratin 19; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPO: myeloperoxidase; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; ND: normal donor; NQO1: NAD(P)H quinone dehydrogenase 1; PCNA: proliferating cell nuclear antigen; RIPA: radio-immunoprecipitation; SQSTM1/p62: sequestosome 1; SOX9: SRY-box transcription factor 9; TAP: trypsinogen activation peptide; TFEB: transcription factor EB; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; UB: ubiquitin; VMP1: vacuole membrane protein 1; XBP1: X-box binding protein 1; YAP1, Yes1 associated transcriptional regulator; ZG: zymogen granule.
    Keywords:  Autophagy; ER stress; Nrf2; oxidative stress; p62
  2. J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01145-5. [Epub ahead of print] 101339
      Mitochondria are important organelles in eukaryotes. Turnover and quality control of mitochondria are regulated at the transcriptional and post-translational level by several cellular mechanisms. Removal of defective mitochondrial proteins is mediated by mitochondria resident proteases or by proteasomal degradation of individual proteins. Clearance of bulk mitochondria occurs via a selective form of autophagy termed mitophagy. In yeast and some developing metazoan cells (e.g. oocytes and reticulocytes), mitochondria are largely removed by ubiquitin-independent mechanisms. In such cases the regulation of mitophagy is mediated via phosphorylation of mitochondria-anchored autophagy receptors. On the other hand, ubiquitin-dependent recruitment of cytosolic autophagy receptors occurs in situations of cellular stress or disease, where dysfunctional mitochondria would cause oxidative damage. In mammalian cells, a well-studied ubiquitin-dependent mitophagy pathway induced by mitochondrial depolarization is regulated by the mitochondrial protein kinase PINK1 that upon activation recruits the ubiquitin ligase parkin. Here we review mechanisms of mitophagy with an emphasis on post-translational modifications that regulate various mitophagy pathways. We describe the autophagy components involved with particular emphasis on post-translational modifications. We detail the phosphorylations mediated by PINK1 and parkin-mediated ubiquitylations of mitochondrial proteins that can be modulated by deubiquitylating enzymes. We also discuss the role of accessory factors regulating mitochondrial fission/fusion and the interplay with pro- and anti-apoptotic Bcl-2 family members. Comprehensive knowledge of the processes of mitophagy is essential for the understanding of vital mitochondrial turnover in health and disease.
    Keywords:  autophagy; mitochondria; phosphorylation; protein kinase PINK1; ubiquitin ligase parkin; ubiquitylation
  3. Sci Rep. 2021 Oct 26. 11(1): 21134
      The sarcomere protein titin is a major determinant of cardiomyocyte stiffness and ventricular distensibility. The constant mechanical stress on titin requires well-controlled protein quality control, the exact mechanisms of which have not yet been fully elucidated. Here, we analyzed E3-ligases potentially responsible for cardiac titin ubiquitination and specifically studied the involvement of the autophagosomal system in titin degradation. Pharmacological inhibition of autophagy and the proteasome in cultured primary rat cardiomyocytes significantly elevated titin ubiquitination and increased titin degradation. Using in-vitro pull down assays we identified binding of E3-ligases MuRF1-3, CHIP and Fbx32 to several titin domains. Immunofluorescence analysis showed sarcomeric localization of the E3-ligases. siRNA-mediated knock-down of the E3-ligases MuRF-1, -3 and a combination of CHIP/Fbx32 significantly reduced autophagy-related titin ubiquitination, whereas knock-down of MuRF-2 and -3 reduced proteasome-related titin ubiquitination. We demonstrated that the proteasomal and the autophagosomal-lysosomal system participate in degradation of the titin filament. We found that ubiquitination and degradation of titin are partially regulated by E3-ligases of the MuRF family. We further identified CHIP and Fbx32 as E3-ligases involved in titin ubiquitination.
  4. Front Genet. 2021 ;12 770853
      Notch signaling pathway plays crucial roles in animal development. Protein ubiquitination contributes to Notch signaling regulation by governing the stability and activity of major signaling components. Studies in Drosophila have identified multiple ubiquitin ligases and deubiquitinating enzymes that modify Notch ligand and receptor proteins. The fate of ubiquitinated substrates depend on topologies of the attached ubiquitin chains, which are determined by the ubiquitin conjugating enzymes (E2 enzymes). However, which E2 enzymes participate in Notch signal transduction remain elusive. Here, we report that the E2 enzyme UbcD1 is required for Notch signaling activation during Drosophila wing development. Mutations of UbcD1 lead to marginal nicks in the adult wing and reduction of Notch signaling targets expression in the wing imaginal disc. Genetic analysis reveal that UbcD1 functions in the signaling receiving cells prior to cleavage of the Notch protein. We provide further evidence suggesting that UbcD1 is likely involved in endocytic trafficking of Notch protein. Our results demonstrate that UbcD1 positively regulates Notch signaling and thus reveal a novel role of UbcD1 in development.
    Keywords:  Drosophila; Notch; UbcD1; ubiquitin conjugating enzyme; wing
  5. Cell Rep. 2021 Oct 26. pii: S2211-1247(21)01349-8. [Epub ahead of print]37(4): 109879
      SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage.
    Keywords:  Fanconi anemia; RNF168; SLX4; interstrand crosslink repair; ubiquitination
  6. J Neural Transm (Vienna). 2021 Oct 23.
      Protein homeostasis, or proteostasis, is essential for cell function and viability. Unwanted, damaged, misfolded and aggregated proteins are degraded by the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. Growing evidence indicates that alterations in these major proteolytic mechanisms lead to a demise in proteostasis, contributing to the onset and development of distinct diseases. Indeed, dysregulation of the UPS or autophagy is linked to several neurodegenerative, infectious and inflammatory disorders as well as cancer. Thus, modulation of protein clearance pathways is a promising approach for therapeutics. In this review, we discuss recent findings and open questions on how targeting proteolytic mechanisms could be applied for disease intervention.
    Keywords:  Aging; Alzheimer’s disease; Amyotrophic lateral sclerosis; Autophagy; Cancer; Huntington’s disease; Parkinson’s disease; Proteasome; Proteostasis
  7. Diabetes. 2021 Nov;70(11): 2431-2443
      Type 2 diabetes (T2D) is a growing cause of poor health, psychosocial burden, and economic costs worldwide. The pancreatic β-cell is a cornerstone of metabolic physiology. Insulin deficiency leads to hyperglycemia, which was fatal before the availability of therapeutic insulins; even partial deficiency of insulin leads to diabetes in the context of insulin resistance. Comprising only an estimated 1 g or <1/500th of a percent of the human body mass, pancreatic β-cells of the islets of Langerhans are a vulnerable link in metabolism. Proinsulin production constitutes a major load on β-cell endoplasmic reticulum (ER), and decompensated ER stress is a cause of β-cell failure and loss in both type 1 diabetes (T1D) and T2D. The unfolded protein response (UPR), the principal ER stress response system, is critical for maintenance of β-cell health. Successful UPR guides expansion of ER protein folding capacity and increased β-cell number through survival pathways and cell replication. However, in some cases the ER stress response can cause collateral β-cell damage and may even contribute to diabetes pathogenesis. Here we review the known beneficial and harmful effects of UPR pathways in pancreatic β-cells. Improved understanding of this stress response tipping point may lead to approaches to maintain β-cell health and function.
  8. Cell Tissue Res. 2021 Oct 29.
      In insects, the follicle cells (FCs) give rise to a single-layered tissue of binucleated professional secretory cells that surround the oocytes during oogenesis. In the latest stage of oocyte development, the FCs rapidly synthesize and secrete the chorion (eggshell) immediately before degenerating through apoptosis. Here, we used RT-qPCR, electron microscopy, and RNAi silencing to explore the role of the main unfolded protein response (UPR) receptors IRE1 and PERK, as well as the ultrastructure dynamics of the FCs during oogenesis of the insect vector of Chagas disease Rhodnius prolixus. We found that IRE1 and PERK mRNAs are highly expressed in the ovaries of vitellogenic females. Interestingly, we observed that IRE1 and PERK, as well as different isoforms of the chaperones Bip and PDI, have their FCs gene expression levels decreased during the vitellogenesis to choriogenesis transition. Using transmission electron microscopy, we observed that the downregulation of the UPR gene expression is accompanied by dramatic changes in the FCs ultrastructure, with an 80% reduction in the mean area of the ER tubules, and circularization and enlargement of the mitochondria. Additionally, we found that parental RNAi silencing of both IRE1 and PERK resulted in minor changes in the chorion protein composition and ultrastructure, accessed by urea extraction of the chorion proteins and scanning electron microscopy, respectively, but did not impact the overall levels of oviposition and F1 embryo development.
    Keywords:  Choriogenesis; Endoplasmic reticulum; Follicle cells; IRE1; PERK
  9. EMBO Rep. 2021 Oct 26. e53429
      Selective autophagy of damaged organelles is important to maintain cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here, we identify two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Autophagy of early endosomes damaged by chloroquine or monensin treatment requires Rabaptin5 and particularly a short sequence motif that binds to the WD domain of ATG16L1. Rabaptin5 and its interaction with ATG16L1 further contributes to the autophagic elimination of Salmonella enterica early after infection, when it resides in phagosomes with early endosomal characteristics. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective targeting of autophagy to damaged early endosomes and phagosomes.
    Keywords:  ATG16L1; Rabaptin5; Salmonella-containing vacuoles; autophagy; early endosomes
  10. Mol Biol Cell. 2021 Oct 27. mbcE21050229
      Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are upregulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited upon RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.
  11. Nat Rev Drug Discov. 2021 Oct 26.
      The accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress, resulting in activation of the unfolded protein response (UPR) that aims to restore protein homeostasis. However, the UPR also plays an important pathological role in many diseases, including metabolic disorders, cancer and neurological disorders. Over the last decade, significant effort has been invested in targeting signalling proteins involved in the UPR and an array of drug-like molecules is now available. However, these molecules have limitations, the understanding of which is crucial for their development into therapies. Here, we critically review the existing ER stress and UPR-directed drug-like molecules, highlighting both their value and their limitations.
  12. Data Brief. 2021 Dec;39 107471
      EDEM2 (Endoplasmic reticulum Degradation-Enhancing alpha-Mannosidase-like protein 2) is one of the key-proteins suggested to be involved in the selection and degradation of misfolded proteins from the endoplasmic reticulum. The datasets discussed in this article are related to experiments covering affinity proteomics, label-free quantitative proteomics, deglycoproteomics and SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) proteomics data of A375 melanoma cells with modified expression of EDEM2. Our first aim was to affinity-enrich EDEM2 alongside its potential interaction partners and analyse the obtained samples by nanoLC-MS/MS to identify novel EDEM2 associated proteins. The dataset was substantiated by SDF (Sucrose Density Fractionation)-nanoLC-MS/MS experiments, in an integrated workflow to validate EDEM2 identified partners and corroborate these with previous data. Our second aim was to delineate novel EDEM2 substrate candidates using a two-step strategy. The first one refers to the deglycoproteomics dataset, which covers nanoLC-MS/MS analysis of Concanavalin A enriched glycopeptides released by endoglycosidase digestion from A375 melanoma cell lysates. This allowed us to map the fraction of glycoproteins with non-matured N-glycans from A375 melanoma cells and find or validate N-glycosylation sites of proteins from the secretory pathway. The same dataset was also used to define glycoproteins altered by the down-regulation of endogenous EDEM2, which should contain its candidate-substrates. In a second step we delineate the degradation kinetics of some of these proteins using a pulse SILAC strategy (pSILAC) thus complementing our initial findings with a fourth dataset. Beside nanoLC-MS/MS analysis our findings were also validated by various biochemical experiments. All the data described are associated with a research article published in Molecular and Cellular Proteomics [1].
    Keywords:  Cancer; Endoplasmic reticulum associated degradation (ERAD); ITGA1; Mass spectrometry; Proteasome; Stable isotope labeling by amino acids in cell culture (SILAC); Tyrosinase; liquid chromatography
  13. Mol Cell. 2021 Oct 15. pii: S1097-2765(21)00800-5. [Epub ahead of print]
      Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.
    Keywords:  autophagy; iNeurons; mitophagy; proteomics
  14. Front Cell Dev Biol. 2021 ;9 710247
      Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, β-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation.
    Keywords:  DNAJC3; cholesterol-stress; mitochondria; proteomics; unfolded protein response (UPR)
  15. Biochem J. 2021 Oct 27. pii: BCJ20210508. [Epub ahead of print]
      Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterization of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 in the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared to wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.
    Keywords:  Parkinsons disease; USP30; USP30 inhibitors; mitoKeima; mitophagy; p-Ser65-ubiquitin
  16. Proc Natl Acad Sci U S A. 2021 Nov 02. pii: e2114258118. [Epub ahead of print]118(44):
      Estrogen receptor α (ER-α) mediates estrogen-dependent cancer progression and is expressed in most breast cancer cells. However, the molecular mechanisms underlying the regulation of the cellular abundance and activity of ER-α remain unclear. We here show that the protein phosphatase calcineurin regulates both ER-α stability and activity in human breast cancer cells. Calcineurin depletion or inhibition down-regulated the abundance of ER-α by promoting its polyubiquitination and degradation. Calcineurin inhibition also promoted the binding of ER-α to the E3 ubiquitin ligase E6AP, and calcineurin mediated the dephosphorylation of ER-α at Ser294 in vitro. Moreover, the ER-α (S294A) mutant was more stable and activated the expression of ER-α target genes to a greater extent compared with the wild-type protein, whereas the extents of its interaction with E6AP and polyubiquitination were attenuated. These results suggest that the phosphorylation of ER-α at Ser294 promotes its binding to E6AP and consequent degradation. Calcineurin was also found to be required for the phosphorylation of ER-α at Ser118 by mechanistic target of rapamycin complex 1 and the consequent activation of ER-α in response to β-estradiol treatment. Our study thus indicates that calcineurin controls both the stability and activity of ER-α by regulating its phosphorylation at Ser294 and Ser118 Finally, the expression of the calcineurin A-α gene (PPP3CA) was associated with poor prognosis in ER-α-positive breast cancer patients treated with tamoxifen or other endocrine therapeutic agents. Calcineurin is thus a promising target for the development of therapies for ER-α-positive breast cancer.
    Keywords:  breast cancer; calcineurin; estrogen receptorα; ubiquitination
  17. Viruses. 2021 Oct 08. pii: 2029. [Epub ahead of print]13(10):
      Zika virus (ZIKV) is a mosquito-borne flavivirus, and its infection may cause severe neurodegenerative diseases. The outbreak of ZIKV in 2015 in South America has caused severe human congenital and neurologic disorders. Thus, it is vitally important to determine the inner mechanism of ZIKV infection. Here, our data suggested that the ubiquitin-specific peptidase 38 (USP38) played an important role in host resistance to ZIKV infection, during which ZIKV infection did not affect USP38 expression. Mechanistically, USP38 bound to the ZIKV envelope (E) protein through its C-terminal domain and attenuated its K48-linked and K63-linked polyubiquitination, thereby repressed the infection of ZIKV. In addition, we found that the deubiquitinase activity of USP38 was essential to inhibit ZIKV infection, and the mutant that lacked the deubiquitinase activity of USP38 lost the ability to inhibit infection. In conclusion, we found a novel host protein USP38 against ZIKV infection, and this may represent a potential therapeutic target for the treatment and prevention of ZIKV infection.
    Keywords:  USP38; Zika virus; deubiquitinase; envelope protein; virus infection
  18. EMBO Rep. 2021 Oct 26. e52509
      Aneuploidy is a chromosomal abnormality associated with poor prognosis in many cancer types. Here, we tested the hypothesis that the unfolded protein response (UPR) mechanistically links aneuploidy and local immune dysregulation. Using a single somatic copy number alteration (SCNA) score inclusive of whole-chromosome, chromosome arm, and focal alterations in a pan-cancer analysis of 9,375 samples in The Cancer Genome Atlas (TCGA) database, we found an inverse correlation with a cytotoxicity (CYT) score across disease stages. Co-expression patterns of UPR genes changed substantially between SCNAlow and SCNAhigh groups. Pathway activity scores showed increased activity of multiple branches of the UPR in response to aneuploidy. The PERK branch showed the strongest association with a reduced CYT score. The conditioned medium of aneuploid cells transmitted XBP1 splicing and caused IL-6 and arginase 1 transcription in receiver bone marrow-derived macrophages and markedly diminished the production of IFN-γ and granzyme B in activated human T cells. We propose the UPR as a mechanistic link between aneuploidy and immune dysregulation in the tumor microenvironment.
    Keywords:  T cells; aneuploidy; macrophages; tumor immune microenvironment; unfolded protein response
  19. J Biol Chem. 2021 Oct 21. pii: S0021-9258(21)01138-8. [Epub ahead of print] 101332
      Embryonic stem cells (ESCs) are progenitor cells that retain the ability to differentiate into various cell types and are necessary for tissue repair. Improving cell culture conditions to maintain the pluripotency of ESCs in vitro is an urgent problem in the field of regenerative medicine. Here, we reveal that Spautin-1, a specific small molecule inhibitor of ubiquitin-specific protease (USP) family members USP10 and USP13, promotes the maintenance of self-renewal and pluripotency of mouse ESCs in vitro. Functional studies reveal that only knockdown of USP13, but not USP10, is capable of mimicking the function of Spautin-1. Mechanistically, we demonstrate that USP13 physically interacts with, deubiquitinates, and stabilizes serine/threonine kinase Raf1 and thereby sustains Raf1 protein at the posttranslational level to activate the FGF/MEK/ERK pro-differentiation signaling pathway in naïve mouse ESCs. In contrast, in primed mouse epiblast stem cells and human induced-pluripotent stem cells, the addition of Spautin-1 had an inhibitory effect on Raf1 levels, but USP13 overexpression promoted self-renewal. The addition of a MEK inhibitor impaired the effect of USP13 upregulation in these cells. These findings provide new insights into the regulatory network of naïve and primed pluripotency as a result of Raf1 deubiquitylation.
    Keywords:  Embryonic stem cells; Raf1; Self-renewal; Spautin-1; USP13
  20. Cell Rep. 2021 Oct 26. pii: S2211-1247(21)01371-1. [Epub ahead of print]37(4): 109901
      The Wnt family contains conserved secretory proteins required for developmental patterning and tissue homeostasis. However, how Wnt is targeted to the endoplasmic reticulum (ER) for processing and secretion remains poorly understood. Here, we report that CATP-8/P5A ATPase directs neuronal migration non-cell autonomously in Caenorhabditis elegans by regulating EGL-20/Wnt biogenesis. CATP-8 likely functions as a translocase to translocate nascent EGL-20/Wnt polypeptide into the ER by interacting with the highly hydrophobic core region of EGL-20 signal sequence. Such regulation of Wnt biogenesis by P5A ATPase is common in C. elegans and conserved in human cells. These findings describe the physiological roles of P5A ATPase in neural development and identify Wnt proteins as direct substrates of P5A ATPase for ER translocation.
    Keywords:  C. elegans; ER translocation; P5A ATPase; Wnt signaling; neuronal migration; signal sequence
  21. Cell Rep. 2021 Oct 26. pii: S2211-1247(21)01374-7. [Epub ahead of print]37(4): 109904
      Inflammasomes are crucial for innate immunity against infections and, when deregulated, also contribute to inflammatory diseases. Here, we identify a critical function of the E3 ubiquitin ligase Peli1 in regulating the activation of NLRP3 inflammasome. Peli1 deficiency impairs induction of interleukin-1β (IL-1β) secretion by different NLRP3 inducers, but not by inducers of the Aim2, NLRP1, and NLRC4 inflammasomes. Peli1-deficient mice have alleviated peritonitis induction by alum and display increased resistance to lipopolysaccharide (LPS) endotoxin shock, coupled with decreased serum concentration of IL-1β. Peli1 is required for NLRP3-induced caspase-1 activation and IL-1β maturation. Mechanistically, Peli1 conjugates K63 ubiquitin chain to lysine 55 of the inflammasome adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which in turn facilitates ASC/NLRP3 interaction and ASC oligomerization, thereby contributing to inflammasome activation. Peli1 deficiency impairs the ubiquitination of ASC and inhibits inflammasome activation. Our findings establish Peli1 as an important inflammasome regulator and suggest a mechanism by which Peli1 mediates inflammatory responses.
    Keywords:  ASC; Peli1; inflammasome; ubiquitination
  22. Elife. 2021 Oct 26. pii: e70357. [Epub ahead of print]10
      Sequential mannose trimming of N-glycan, from M9 to M8B and then to oligosaccharides exposing the a1,6-linked mannosyl residue (M7A, M6 and M5), facilitates endoplasmic reticulum-associated degradation of misfolded glycoproteins (gpERAD). We previously showed that EDEM2 stably disulfide-bonded to the thioredoxin domain-containing protein TXNDC11 is responsible for the first step (George et al., 2020). Here, we show that EDEM3 and EDEM1 are responsible for the second step. Incubation of pyridylamine-labeled M8B with purified EDEM3 alone produced M7 (M7A and M7C), M6 and M5. EDEM1 showed a similar tendency, although much lower amounts of M6 and M5 were produced. Thus, EDEM3 is a major a1,2-mannosidase for the second step from M8B. Both EDEM3 and EDEM1 trimmed M8B from a glycoprotein efficiently. Our confirmation of the Golgi localization of MAN1B indicates that no other a1,2-mannosidase is required for gpERAD. Accordingly, we have established the entire route of oligosaccharide processing and the enzymes responsible.
    Keywords:  biochemistry; cell biology; chemical biology; human
  23. Nat Commun. 2021 Oct 26. 12(1): 6173
      The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.
  24. J Cell Biol. 2021 Dec 06. pii: e202103030. [Epub ahead of print]220(12):
      Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.
  25. Dev Cell. 2021 Oct 22. pii: S1534-5807(21)00809-1. [Epub ahead of print]
      In order to combat molecular damage, most cellular proteins undergo rapid turnover. We have previously identified large nuclear protein assemblies that can persist for years in post-mitotic tissues and are subject to age-related decline. Here, we report that mitochondria can be long lived in the mouse brain and reveal that specific mitochondrial proteins have half-lives longer than the average proteome. These mitochondrial long-lived proteins (mitoLLPs) are core components of the electron transport chain (ETC) and display increased longevity in respiratory supercomplexes. We find that COX7C, a mitoLLP that forms a stable contact site between complexes I and IV, is required for complex IV and supercomplex assembly. Remarkably, even upon depletion of COX7C transcripts, ETC function is maintained for days, effectively uncoupling mitochondrial function from ongoing transcription of its mitoLLPs. Our results suggest that modulating protein longevity within the ETC is critical for mitochondrial proteome maintenance and the robustness of mitochondrial function.
    Keywords:  age mosaicism; aging; electron transport chain; heterogeneity; long-lived proteins; mitochondria; muscle; neurons; protein homeostasis; supercomplexes
  26. Oncogene. 2021 Oct 29.
      ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.
  27. Elife. 2021 Oct 27. pii: e65759. [Epub ahead of print]10
      RNF43 is an E3 ubiquitin ligase and known negative regulator of WNT/β-catenin signaling. We demonstrate that RNF43 is also a regulator of noncanonical WNT5A-induced signaling in human cells. Analysis of the RNF43 interactome using BioID and immunoprecipitation showed that RNF43 can interact with the core receptor complex components dedicated to the noncanonical Wnt pathway such as ROR1, ROR2, VANGL1, and VANGL2. RNF43 triggers VANGL2 ubiquitination and proteasomal degradation and clathrin-dependent internalization of ROR1 receptor and inhibits ROR2 activation. These activities of RNF43 are physiologically relevant and block pro-metastatic WNT5A signaling in melanoma. RNF43 inhibits responses to WNT5A, which results in the suppression of invasive properties of melanoma cells. Furthermore, RNF43 prevented WNT5A-assisted development of resistance to BRAF V600E and MEK inhibitors. Next, RNF43 acted as melanoma suppressor and improved response to targeted therapies in vivo. In line with these findings, RNF43 expression decreases during melanoma progression and RNF43-low patients have a worse prognosis. We conclude that RNF43 is a newly discovered negative regulator of WNT5A-mediated biological responses that desensitizes cells to WNT5A.
    Keywords:  BRAF V600E; RNF43; ROR1; VANGL1; WNT5A; cancer biology; cell biology; human; melanoma; mouse
  28. Nature. 2021 Oct 28.
      Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1-3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes.