bims-proteo Biomed News
on Proteostasis
Issue of 2021‒09‒12
38 papers selected by
Eric Chevet

  1. BMB Rep. 2021 Sep 07. pii: 5414. [Epub ahead of print]
      Translating ribosomes accompany co-translational regulation of nascent polypeptide chains, including subcellular targeting, protein folding, and covalent modifications. Ribosome-associated quality control (RQC) is a co-translational surveillance mechanism triggered by ribosomal collisions, an indication of atypical translation. The ribosome-associated E3 ligase ZNF598 ubiquitinates small subunit proteins at the stalled ribosomes. A series of RQC factors are then recruited to dissociate and triage aberrant translation intermediates. Regulatory ribosomal stalling may occur on endogenous transcripts for quality gene expression, whereas ribosomal collisions are more globally induced by ribotoxic stressors such as translation inhibitors, ribotoxins, and UV radiation. The latter are sensed by ribosome-associated kinases GCN2 and ZAK!, activating integrated stress response (ISR) and ribotoxic stress response (RSR), respectively. Hierarchical crosstalks among RQC, ISR, and RSR pathways are readily detectable since the collided ribosome is their common substrate for activation. Given the strong implications of RQC factors in neuronal physiology and neurological disorders, the interplay between RQC and ribosome-associated stress signaling may sustain proteostasis, adaptively determine cell fate, and contribute to neural pathogenesis. The elucidation of underlying molecular principles in relevant human diseases should thus provide unexplored therapeutic opportunities.
  2. Curr Opin Plant Biol. 2021 Sep 03. pii: S1369-5266(21)00106-0. [Epub ahead of print]63 102106
      Maintaining the integrity of organelles despite the cellular disturbances that arise during stress is essential for life. To ensure organelle proteostasis (protein homeostasis), plants have evolved multitiered quality control mechanisms that work together to repair or recycle the damaged organelles. Despite recent advances, our understanding of plant organelle quality control mechanisms is far from complete. Especially, the crosstalk between different quality control pathways remains elusive. Here, we highlight recent advances on organelle quality control, focusing on the targeted protein degradation pathways that maintain the homeostasis of the endoplasmic reticulum (ER), chloroplast, and mitochondria. We discuss how plant cells decide to employ different degradation pathways and propose tools that could be used to discover the missing components in organelle quality control.
    Keywords:  CDC48; Model substrates; Organelle quality control; Proteasome; Selective autophagy; Unfolded protein response; Viral replication
  3. Autophagy. 2021 Sep 07. 1-3
      Clearance of misfolded proteins from the secretory pathway often occurs soon after their biosynthesis by endoplasmic reticulum (ER)-associated protein degradation (ERAD). However, certain types of misfolded proteins are not ERAD substrates and exit the ER. They are then scrutinized by ill-defined post-ER quality control (post-ERQC) mechanisms and are frequently routed to the vacuole/lysosome for degradation. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) constitute a class of proteins of the secretory pathway that mostly depends on post-ERQC. How misfolded GPI-APs are detected, transported to the vacuole/lysosome and taken up by this organelle was poorly defined. Originating from the intriguing observation that several misfolded GPI-APs accumulate in the yeast vacuolar membrane in the absence of the major vacuolar protease Pep4, we designed an unbiased genome-wide screen in yeast and followed the trafficking of the misfolded fluorescent GPI-AP Gas1* from the ER to the vacuolar lumen. Our results reveal that post-ERQC of GPI-APs is linked with a novel type of microautophagy.
  4. Int J Mol Sci. 2021 Sep 06. pii: 9655. [Epub ahead of print]22(17):
      Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1) and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation (ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the current knowledge about the cooperation of the ER and mitochondria in the targeting and quality control of mitochondrial precursor proteins.
    Keywords:  ER-SURF; chaperones; contact sites; endoplasmic reticulum; membrane extraction; mitochondria; protein targeting
  5. FEBS J. 2021 Sep 07.
      The endoplasmic reticulum (ER) is equipped with multiple quality control systems that are necessary for shaping the glycoproteome of eukaryotic cells. These systems facilitate the productive folding of glycoproteins, eliminate defective products, and function as effectors to evoke cellular signaling in response to various cellular stresses. These ER functions largely depend on glycans, which contain sugar-based codes that, when needed, function to recruit carbohydrate-binding proteins that determine the fate of glycoproteins. To ensure their functionality, the biosynthesis of such glycans is therefore strictly monitored by a system that selectively degrades structurally defective glycans before adding them to proteins. This system, which is referred to as the glycan quality control system (QCS), serves as a mechanism to reduce the risk of abnormal glycosylation under conditions where glycan biosynthesis is genetically or metabolically stalled. On the other hand, glycan QCS increases the risk of global hypoglycosylation by limiting glycan availability, which can lead to protein misfolding and the activation of unfolded protein response to maintaining cell viability or to initiate cell death programs. This review summarizes the current state of our knowledge of the mechanisms underlying glycan QCS in mammals and its physiological and pathological roles in embryogenesis, tumor progression and congenital disorders associated with abnormal glycosylation.
    Keywords:  Asparagine-linked glycosylation; carbohydrate metabolism; congenital disorders of glycosylation; dolichol-linked oligosaccharides; glycoproteins; nucleotide sugars; quality control
  6. Cell Chem Biol. 2021 Sep 07. pii: S2451-9456(21)00396-2. [Epub ahead of print]
      While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.
    Keywords:  E3 ligase; JQ1; SKP1; SOCS box family; SPSB2; VHL; covalent; dasatinib; electroporation; neo-substrate; targeted protein degradation (TPD)
  7. FASEB J. 2021 Oct;35(10): e21865
      Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.
    Keywords:  ADPKD; GRP94; HDAC6; HSP; HSP90; UPR; autophagy; chaperone; kidney; polycystin-1; polycystin-2; proteasome
  8. J Biol Chem. 2021 Sep 04. pii: S0021-9258(21)00973-X. [Epub ahead of print] 101171
      The yeast endoplasmic reticulum has three distinct protein translocation channels. The heterotrimeric Sec61 and Ssh1 complexes, which bind translating ribosomes, mediate cotranslational translocation of proteins targeted to the endoplasmic reticulum by the signal recognition particle (SRP) and SRP receptor targeting pathway, whereas the heptameric Sec complex has been proposed to mediate ribosome-independent posttranslational translocation of proteins with less hydrophobic signal sequences that escape recognition by the SRP. However, multiple reports have proposed that the Sec complex may function cotranslationally and be involved in translocation or integration of SRP-dependent protein translocation substrates. To provide insight into these conflicting views, we induced expression of the tobacco etch virus (TEV) protease to achieve rapid inactivation of the Sec complex by protease-mediated cleavage within the cytoplasmic domain of the Sec63 protein. Protein translocation assays conducted after TEV protease induction revealed a complete block in translocation of two well-characterized substrates of the Sec complex, carboxypeptidase Y (CPY) and Gas1p, when the protease cleavage sites were located at structural domain boundaries in Sec63. However, integration of SRP-dependent membrane protein substrates was not detectably impacted. Moreover, redirecting CPY to the cotranslational pathway by increasing the hydrophobicity of the signal sequence rendered translocation of CPY insensitive to inactivation of the Sec complex. We conclude that the Sec complex is primarily responsible for the translocation of yeast secretome proteins with marginally hydrophobic signal sequences.
    Keywords:  endoplasmic reticulum (ER); membrane protein; protein synthesis; protein translocation; ribosome
  9. Nat Commun. 2021 Sep 09. 12(1): 5354
      Mitochondrial division is not an autonomous event but involves multiple organelles, including the endoplasmic reticulum (ER) and lysosomes. Whereas the ER drives the constriction of mitochondrial membranes, the role of lysosomes in mitochondrial division is not known. Here, using super-resolution live-cell imaging, we investigate the recruitment of lysosomes to the site of mitochondrial division. We find that the ER recruits lysosomes to the site of division through the interaction of VAMP-associated proteins (VAPs) with the lysosomal lipid transfer protein ORP1L to induce a three-way contact between the ER, lysosome, and the mitochondrion. We also show that ORP1L might transport phosphatidylinositol-4-phosphate (PI(4)P) from lysosomes to mitochondria, as inhibiting its transfer or depleting PI(4)P at the mitochondrial division site impairs fission, demonstrating a direct role for PI(4)P in the division process. Our findings support a model where the ER recruits lysosomes to act in concert at the fission site for the efficient division of mitochondria.
  10. Chembiochem. 2021 Sep 08.
      Proteolysis-targeting chimeras (PROTACs), an emerging paradigm-shifting technology, hijacks the ubiquitin-proteasome system for targeted protein degradation. PROTACs induce ternary complexes between an E3 ligase and protein of interest (POI), and this induced proximity leads to poly-ubiquitin chain formation on substrate proteins and eventual proteasomal-mediated POI degradation. PROTACs have shown therapeutic potential by degrading many disease-causing proteins, such as the androgen receptor and BRD4. The PROTAC technology has advanced significantly in the last two decades, targeting an expanding repertoire of POIs. Herein, we describe recent PROTAC technology developments, focusing on mechanistic and kinetic studies, pharmacokinetic studies, spatiotemporal control of PROTACs, covalent PROTACs, resistance to PROTACs, and new E3 ligands.
    Keywords:  PROTACs, Targeted Protein Degradation, Ligases, Ubiquitin, Proteasome
  11. iScience. 2021 Sep 24. 24(9): 102985
      Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand.
    Keywords:  biological sciences; cell biology; molecular biology; molecular mechanism of gene regulation; transcriptomics
  12. Nat Cell Biol. 2021 Sep 08.
      The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif-containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.
  13. Autophagy. 2021 Sep 05. 1-15
      ABBREVIATIONS: 3-MA: 3-methyladenine; AIM2: absent in melanoma 2; ATG5: autophagy related 5; BafA1: bafilomycin A1; CASP1: caspase 1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CQ: chloroquine; DUBs: deubiquitinases; IL1B/IL-1β: interleukin 1 beta; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MARCHF7/MARCH7: membrane associated RING-CH-type finger 7; NFKB/NF-κB: nuclear factor kappa B; Nig.: nigericin; NLRC4: NLR family CARD domain containing 4; NLRP3: NLR family pyrin domain containing 3; PECs: peritoneal exudate cells; PMN: polymorphonuclear; PMs: peritoneal macrophages; PYCARD/ASC: PYD and CARD domain containing; TLRs: toll like receptors; TNF/TNF-α: tumor necrosis factor; Ub: ubiquitin; USP5: ubiquitin specific peptidase 5; WT: wild type.
    Keywords:  Autophagy-lysosome pathway; MARCHF7; NLRP3 inflammasome; USP5; deubiquitinase
  14. Mol Cell Proteomics. 2021 Sep 02. pii: S1535-9476(21)00116-X. [Epub ahead of print] 100144
      Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of type I integral membrane protein human CD4 (huCD4) across the ER in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared to huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for these proteins, we employed stable isotope labelling by amino acids in cell culture (SILAC) technique in combination with quantitative mass spectrometry on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA-treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, Endoplasmic Reticulum Lectin 1 (ERLEC1), Inactive Tyrosine-Protein Kinase 7 (PTK7), and DnaJ Homolog Subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7 and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable signal peptides that are targets for the co-translational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7 and DNAJC3 are validated additional substrates of CADA, however, huCD4 remains the most sensitive integral membrane protein for the ER translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound which specifically interferes with only a very small subset of signal peptides and does not affect signal anchor sequences.
    Keywords:  CD4; ER co-translational translocation; SILAC; cyclotriazadisulfonamide (CADA); signal peptide; small molecule inhibitor
  15. BMC Bioinformatics. 2021 Sep 08. 22(1): 427
      BACKGROUND: In mammalian cells the endoplasmic reticulum (ER) comprises a highly complex reticular morphology that is spread throughout the cytoplasm. This organelle is of particular interest to biologists, as its dysfunction is associated with numerous diseases, which often manifest themselves as changes to the structure and organisation of the reticular network. Due to its complex morphology, image analysis methods to quantitatively describe this organelle, and importantly any changes to it, are lacking.RESULTS: In this work we detail a methodological approach that utilises automated high-content screening microscopy to capture images of cells fluorescently-labelled for various ER markers, followed by their quantitative analysis. We propose that two key metrics, namely the area of dense ER and the area of polygonal regions in between the reticular elements, together provide a basis for measuring the quantities of rough and smooth ER, respectively. We demonstrate that a number of different pharmacological perturbations to the ER can be quantitatively measured and compared in our automated image analysis pipeline. Furthermore, we show that this method can be implemented in both commercial and open-access image analysis software with comparable results.
    CONCLUSIONS: We propose that this method has the potential to be applied in the context of large-scale genetic and chemical perturbations to assess the organisation of the ER in adherent cell cultures.
    Keywords:  Automated image analysis; ER function; Endoplasmic reticulum morphology; High-content imaging
  16. Front Immunol. 2021 ;12 695331
      Cullin-RING ligases (CRLs) are a significant subset of Ubiquitin E3 ligases that regulate multiple cellular substrates involved in innate immunity, cytoskeleton modeling, and cell cycle. The glutamine deamidase Cycle inhibitory factor (Cif) from enteric bacteria inactivates CRLs to modulate these processes in the host cell. The covalent attachment of a Ubiquitin-like protein NEDD8 catalytically activates CRLs by driving conformational changes in the Cullin C-terminal domain (CTD). NEDDylation results in a shift from a compact to an open CTD conformation through non-covalent interactions between NEDD8 and the WHB subdomain of CTD, eliminating the latter's inhibitory interactions with the RING E3 ligase-Rbx1/2. It is unknown whether the non-covalent interactions are sufficient to stabilize Cullin CTD's catalytic conformation. We studied the dynamics of Cullin-CTD in the presence and absence of NEDD8 using atomistic molecular dynamics (MD) simulations. We uncovered that NEDD8 engages in non-covalent interactions with 4HB/αβ subdomains in Cullin-CTD to promote open conformations. Cif deamidates glutamine 40 in NEDD8 to inhibit the conformational change in CRLs by an unknown mechanism. We investigated the effect of glutamine deamidation on NEDD8 and its interaction with the WHB subdomain post-NEDDylation using MD simulations and NMR spectroscopy. Our results suggest that deamidation creates a new intramolecular salt bridge in NEDD8 to destabilize the NEDD8/WHB complex and reduce CRL activity.
    Keywords:  Cullin RING E3 ligases; NMR spectroscopy; bacterial effector; cycle inhibitory factor; deamidation; enteropathogenic E. coli; protein dynamics (molecular dynamics)
  17. Biochim Biophys Acta Mol Basis Dis. 2021 Sep 01. pii: S0925-4439(21)00195-2. [Epub ahead of print] 166262
      Autophagy refers to a ubiquitous set of catabolic pathways required to achieve proper cellular homeostasis. Aberrant autophagy has been implicated in a multitude of diseases including cancer. In this review, we highlight pioneering and groundbreaking research that centers on delineating the role of autophagy in cancer initiation, proliferation and metastasis. First, we discuss the autophagy-related (ATG) proteins and their respective roles in the de novo formation of autophagosomes and the subsequent delivery of cargo to the lysosome for recycling. Next, we touch upon the history of cancer research that centers upon ATG proteins and regulatory mechanisms that control an appropriate autophagic response and how these are altered in the diseased state. Then, we discuss the various discoveries that led to the idea of autophagy as a double-edged sword when it comes to cancer therapy. This review also briefly narrates how different types of autophagy-selective macroautophagy and chaperone-mediated autophagy, have been linked to different cancers. Overall, these studies build upon a steadfast trajectory that aims to solve the monumentally daunting challenge of finding a cure for many types of cancer by modulating autophagy either through inhibition or induction.
    Keywords:  Autophagy; cancer; chaperone-mediated autophagy; history; selective autophagy; treatment
  18. Nat Commun. 2021 Sep 07. 12(1): 5321
      CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers.
  19. Nucleic Acids Res. 2021 Sep 09. pii: gkab766. [Epub ahead of print]
      Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that provide the ribosome with aminoacyl-tRNA substrates for protein synthesis. Mutations in aaRSs lead to various neurological disorders in humans. Many aaRSs utilize editing to prevent error propagation during translation. Editing defects in alanyl-tRNA synthetase (AlaRS) cause neurodegeneration and cardioproteinopathy in mice and are associated with microcephaly in human patients. The cellular impact of AlaRS editing deficiency in eukaryotes remains unclear. Here we use yeast as a model organism to systematically investigate the physiological role of AlaRS editing. Our RNA sequencing and quantitative proteomics results reveal that AlaRS editing defects surprisingly activate the general amino acid control pathway and attenuate the heatshock response. We have confirmed these results with reporter and growth assays. In addition, AlaRS editing defects downregulate carbon metabolism and attenuate protein synthesis. Supplying yeast cells with extra carbon source partially rescues the heat sensitivity caused by AlaRS editing deficiency. These findings are in stark contrast with the cellular effects caused by editing deficiency in other aaRSs. Our study therefore highlights the idiosyncratic role of AlaRS editing compared with other aaRSs and provides a model for the physiological impact caused by the lack of AlaRS editing.
  20. Commun Biol. 2021 Sep 06. 4(1): 1038
      Mechanosensitive channels are integral membrane proteins that sense mechanical stimuli. Like most plasma membrane ion channel proteins they must pass through biosynthetic quality control in the endoplasmic reticulum that results in them reaching their destination at the plasma membrane. Here we show that N-linked glycosylation of two highly conserved asparagine residues in the 'cap' region of mechanosensitive Piezo1 channels are necessary for the mature protein to reach the plasma membrane. Both mutation of these asparagines (N2294Q/N2331Q) and treatment with an enzyme that hydrolyses N-linked oligosaccharides (PNGaseF) eliminates the fully glycosylated mature Piezo1 protein. The N-glycans in the cap are a pre-requisite for N-glycosylation in the 'propeller' regions, which are present in loops that are essential for mechanotransduction. Importantly, trafficking-defective Piezo1 variants linked to generalized lymphatic dysplasia and bicuspid aortic valve display reduced fully N-glycosylated Piezo1 protein. Thus the N-linked glycosylation status in vitro correlates with efficient membrane trafficking and will aid in determining the functional impact of Piezo1 variants of unknown significance.
  21. EMBO J. 2021 Sep 06. e108065
      The pyruvate kinase M2 isoform (PKM2) is preferentially expressed in cancer cells to regulate anabolic metabolism. Although PKM2 was recently reported to regulate lipid homeostasis, the molecular mechanism remains unclear. Herein, we discovered an ER transmembrane protein 33 (TMEM33) as a downstream effector of PKM2 that regulates activation of SREBPs and lipid metabolism. Loss of PKM2 leads to up-regulation of TMEM33, which recruits RNF5, an E3 ligase, to promote SREBP-cleavage activating protein (SCAP) degradation. TMEM33 is transcriptionally regulated by nuclear factor erythroid 2-like 1 (NRF1), whose cleavage and activation are controlled by PKM2 levels. Total plasma cholesterol levels are elevated by either treatment with PKM2 tetramer-promoting agent TEPP-46 or by global PKM2 knockout in mice, highlighting the essential function of PKM2 in lipid metabolism. Although depletion of PKM2 decreases cancer cell growth, global PKM2 knockout accelerates allografted tumor growth. Together, our findings reveal the cell-autonomous and systemic effects of PKM2 in lipid homeostasis and carcinogenesis, as well as TMEM33 as a bona fide regulator of lipid metabolism.
    Keywords:  PKM2; SCAP degradation; TMEM33; total cholesterol levels; tumor growth
  22. Oncogenesis. 2021 Sep 10. 10(9): 60
      Tamoxifen resistance remains a clinical problem in estrogen receptor (ER)-positive breast cancer. SUMOylation of ERα enhances ERα-induced transcription activity. Tripartite motif-containing (TRIM) proteins are a new class of SUMO E3 ligases, which regulate the SUMOylation of proteins. However, the precise molecular mechanism and function of TRIM3 in SUMOylation and the response to tamoxifen remain unclear. In the present study, we observed that TRIM3 was dramatically overexpressed in breast cancer, which correlated with tamoxifen resistance. Furthermore, TRIM3 overexpression significantly correlated with poor survival of patients with ER+ breast cancer treated with tamoxifen. TRIM3 overexpression conferred cell survival and tumorigenesis, whereas knocking down of TRIM3 reduced these capabilities. Moreover, TRIM3, as a ubiquitin carrier protein 9 (UBC9) binding protein, promoted SUMO modification of estrogen receptor 1 (ESR1) and activated the ER pathway. Silencing UBC9 abolished the function of TRIM3 in regulating tamoxifen resistance. These results suggest TRIM3 as a novel biomarker for breast cancer therapy, indicating that inhibiting TRIM3 combined with tamoxifen might provide a potential treatment for breast cancer.
  23. Elife. 2021 Sep 06. pii: e69377. [Epub ahead of print]10
      Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions.
    Keywords:  biochemistry; chemical biology; human
  24. J Lipid Res. 2021 Sep 02. pii: S0022-2275(21)00096-1. [Epub ahead of print] 100114
      Niemann Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. These results indicate that manipulation of molecular chaperones may lead to effective treatments for NPC1 disease, but further investigation of mechanisms will be required.
    Keywords:  Cholesterol; Drug therapy; Endocytosis; Fluorescence Microscopy; HSP70; HSP90; Lysosomal Storage; Niemann-Pick disease; arimoclomol; chaperone
  25. RNA. 2021 Sep 08. pii: rna.078954.121. [Epub ahead of print]
      The FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region that are required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.
    Keywords:  Hfq; ProQ; RNA-binding protein; proteolysis; small RNA
  26. Elife. 2021 Sep 10. pii: e70372. [Epub ahead of print]10
      Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed â-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 â-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.
    Keywords:  cell biology; human
  27. Nat Commun. 2021 Sep 09. 12(1): 5338
      Molecular chaperones, including Hsp70/J-domain protein (JDP) families, play central roles in binding substrates to prevent their aggregation. How JDPs select different conformations of substrates remains poorly understood. Here, we report an interaction between the JDP DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a β-turn structural element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau, but not mutant, β-turn structural elements can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. Our work identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.
  28. mBio. 2021 Sep 07. e0193221
      The essential endoribonuclease RNase E, which is a component of the Escherichia coli multienzyme RNA degradosome, has a global role in RNA processing and degradation. RNase E localizes to the inner cytoplasmic membrane in small, short-lived clusters (puncta). Rifampin, which arrests transcription, inhibits RNase E clustering and increases its rate of diffusion. Here, we show that inhibition of clustering is due to the arrest of transcription using a rifampin-resistant control strain. Two components of the RNA degradosome, the 3' exoribonuclease polynucleotide phosphorylase (PNPase) and the DEAD box RNA helicase RhlB, colocalize with RNase E in puncta. Clustering of PNPase and RhlB is inhibited by rifampin, and their diffusion rates increase, as evidenced by in vivo photobleaching measurements. Results with rifampin treatment reported here show that RNA degradosome diffusion is constrained by interaction with RNA substrate. Kasugamycin, which arrests translation initiation, inhibits formation of puncta and increases RNA degradosome diffusion rates. Since kasugamycin treatment results in continued synthesis and turnover of ribosome-free mRNA but inhibits polyribosome formation, RNA degradosome clustering is therefore polyribosome dependent. Chloramphenicol, which arrests translation elongation, results in formation of large clusters (foci) of RNA degradosomes that are distinct from puncta. Since chloramphenicol-treated ribosomes are stable, the formation of RNA degradosome foci could be part of a stress response that protects inactive polyribosomes from degradation. Our results strongly suggest that puncta are sites where translationally active polyribosomes are captured by membrane-associated RNA degradosomes. These sites could be part of a scanning process that is an initial step in mRNA degradation. IMPORTANCE Here, we show that RNase E, RhlB, and PNPase act together as components of the multienzyme RNA degradosome in polyribosome-dependent clustering to form puncta on the inner cytoplasmic membrane. Our results support the hypothesis that RNA degradosome puncta are sites of mRNA degradation. We propose that clustering of RNA degradosomes is a pre-RNase E cleavage step in which polyribosomes are scanned in a search for ribosome-free mRNA. This work is part of an emerging view that posttranscriptional events such as tRNA maturation, late steps in ribosome assembly, and mRNA degradation are membrane associated and partitioned from translation in the cytoplasm and transcription in the nucleoid. This separation could protect newly synthesized transcripts from premature destructive interactions with the RNA degradosome. The scanning of ribosomes and polyribosomes could be part of a general mechanism in which defective stable RNA or ribosome-free mRNA is targeted for destruction by the RNA degradosome.
    Keywords:  RNA degradosome; chloramphenicol; inner cytoplasmic membrane; kasugamycin; mRNA degradation; polyribosome; rifampin
  29. Cell Rep. 2021 Sep 07. pii: S2211-1247(21)01107-4. [Epub ahead of print]36(10): 109663
      Although the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5' poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.
    Keywords:  IRES; alternative initiation; cryo-EM; mRNA specification; post-translational modification; protein synthesis; ribosome; selective translation; structure
  30. Trends Neurosci. 2021 Sep 03. pii: S0166-2236(21)00161-2. [Epub ahead of print]
      Neurons continuously adapt to external cues and challenges, including stimulation, plasticity-inducing signals and aging. These adaptations are critical for neuronal physiology and extended survival. Proteostasis is the process by which cells adjust their protein content to achieve the specific protein repertoire necessary for cellular function. Due to their complex morphology and polarized nature, neurons possess unique proteostatic requirements. Proteostatic control in axons and dendrites must be implemented through regulation of protein synthesis and degradation in a decentralized fashion, but at the same time, it requires integration, at least in part, in the soma. Here, we discuss current understanding of neuronal proteostasis, as well as open questions and future directions requiring further exploration.
    Keywords:  mRNA; post-translational modifications; proteasome; protein degradation; protein synthesis
  31. Nat Commun. 2021 09 06. 12(1): 5263
      Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the β-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.
  32. Structure. 2021 Sep 03. pii: S0969-2126(21)00297-5. [Epub ahead of print]
      R2TP is a highly conserved chaperone complex formed by two AAA+ ATPases, RUVBL1 and RUVBL2, that associate with PIH1D1 and RPAP3 proteins. R2TP acts in promoting macromolecular complex formation. Here, we establish the principles of R2TP assembly. Three distinct RUVBL1/2-based complexes are identified: R2TP, RUVBL1/2-RPAP3 (R2T), and RUVBL1/2-PIH1D1 (R2P). Interestingly, we find that PIH1D1 does not bind to RUVBL1/RUVBL2 in R2TP and does not function as a nucleotide exchange factor; instead, RPAP3 is found to be the central subunit coordinating R2TP architecture and linking PIH1D1 and RUVBL1/2. We also report that RPAP3 contains an intrinsically disordered N-terminal domain mediating interactions with substrates whose sequences are primarily enriched for Armadillo repeat domains and other helical-type domains. Our work provides a clear and consistent model of R2TP complex structure and gives important insights into how a chaperone machine concerned with assembly of folded proteins into multisubunit complexes might work.
    Keywords:  AAA+ proteins; ATPases; PAQosome; R2TP; RUVBL1/2; macromolecular complex assembly; molecular chaperones; protein folding
  33. Am J Physiol Gastrointest Liver Physiol. 2021 Sep 08.
      Goblet cells are specialized for the production and secretion of MUC2 glycoproteins that forms a thick layer covering the mucosal epithelium as a protective barrier against noxious substances and invading microbes. High MUC2 mucin biosynthesis induces endoplasmic reticulum (ER) stress and apoptosis in goblet cells during inflammatory and infectious diseases. Autophagy is an intracellular degradation process required for maintenance of intestinal homeostasis. In this study, we hypothesized that autophagy was triggered during high MUC2 mucin biosynthesis from colonic goblet cells to cope with metabolic stress. To interrogate this, we analyzed the autophagy process in high MUC2-producing human HT29-H and a clone HT29-L silenced for MUC2 expression by lentivirus-mediated shRNA, and WT and CRISPR/Cas9 MUC2 KO LS174T cells. Autophagy was constitutively increased in high MUC2 producing cells characterized by elevated pULK1S555 expression and increased numbers of autophagosomes as compared to MUC2 silenced or gene edited cells. Similarly, colonoids from Muc2+/+ but not Muc2-/-littermates differentiated into goblet cells showed increased autophagy. IL-22 treatment corrected misfolded MUC2 protein and alleviated the autophagy process in LS174T cells. This study highlights that autophagy plays an essential role in goblet cells to survive during high mucin biosynthesis by regulating cellular homeostasis.
    Keywords:  Autophagy; Goblet Cell; Mucin
  34. Br J Cancer. 2021 Sep 08.
      Breast cancer has the highest incidence and death rate among cancers in women worldwide. In particular, metastatic estrogen receptor negative (ER-) breast cancer and triple-negative breast cancer (TNBC) subtypes have very limited treatment options, with low survival rates. Ubiquitin carboxyl terminal hydrolase L1 (UCHL1), a ubiquitin C-terminal hydrolase belonging to the deubiquitinase (DUB) family of enzymes, is highly expressed in these cancer types, and several key reports have revealed emerging and important roles for UCHL1 in breast cancer. However, selective and potent small-molecule UCHL1 inhibitors have been disclosed only very recently, alongside chemical biology approaches to detect regulated UHCL1 activity in cancer cells. These tools will enable novel insights into oncogenic mechanisms driven by UCHL1, and identification of substrate proteins deubiquitinated by UCHL1, with the ultimate goal of realising the potential of UCHL1 as a drug target in breast cancer.
  35. mBio. 2021 Sep 07. e0109721
      The Epstein-Barr virus (EBV) protein LMP1 serves as a paradigm that engages complicated ubiquitination-mediated mechanisms to activate multiple transcription factors. p62 is a ubiquitin sensor and a signal-transducing adaptor that has multiple functions in diverse contexts. However, the interaction between p62 and oncogenic viruses is poorly understood. We recently reported a crucial role for p62 in oncovirus-mediated oxidative stress by acting as a selective autophagy receptor. In this following pursuit, we further discovered that p62 is upregulated in EBV type 3 compared to type 1 latency, with a significant contribution from NF-κB and AP1 activities downstream of LMP1 signaling. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination and activation. As expected, short hairpin RNA (shRNA)-mediated knockdown (KD) of p62 transcripts reduces LMP1-TRAF6 interaction and TRAF6 ubiquitination, as well as p65 nuclear translocation, which was assessed by Amnis imaging flow cytometry. Strikingly, LMP1-stimulated NF-κB, AP1, and Akt activities are all markedly reduced in p62-/- mouse embryo fibroblasts (MEFs) and in EBV-negative Burkitt's lymphoma (BL) cell lines with CRISPR-mediated knockout (KO) of the p62-encoding gene. However, EBV-positive BL cell lines (type 3 latency) with CRISPR-mediated KO of the p62-encoding gene failed to survive. In consequence, shRNA-mediated p62 KD impairs the ability of LMP1 to regulate its target gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of lymphoblastic cell lines (LCLs). These important findings have revealed a previously unrecognized novel role for p62 in EBV latency and oncogenesis, which advances our understanding of the mechanism underlying virus-mediated oncogenesis. IMPORTANCE As a ubiquitin sensor and a signal-transducing adaptor, p62 is crucial for NF-κB activation, which involves the ubiquitin machinery, in diverse contexts. However, whether p62 is required for EBV LMP1 activation of NF-κB is an open question. In this study, we provide evidence that p62 is upregulated in EBV type 3 latency and, in turn, p62 mediates LMP1 signal transduction to NF-κB, AP1, and Akt by promoting TRAF6 ubiquitination and activation. In consequence, p62 deficiency negatively regulates LMP1-mediated gene expression, promotes etoposide-induced apoptosis, and reduces the proliferation of LCLs. These important findings identified p62 as a novel signaling component of the key viral oncogenic signaling pathway.
    Keywords:  EBV; LMP1; herpesviruses; p62; ubiquitination; viral oncogenesis
  36. Hepatology. 2021 May 02.
      BACKGROUND AND AIMS: NAFLD, characterized by aberrant triglyceride accumulation in liver, affects the metabolic remodeling of hepatic and nonhepatic tissues by secreting altered hepatokines. Small ubiquitin-related modifier (SUMO)-specific protease 2 (SENP2) is responsible for de-SUMOylation of target protein, with broad effects on cell growth, signal transduction, and developmental processes. However, the role of SENP2 in hepatic metabolism remains unclear.APPROACH AND RESULTS: We found that SENP2 was the most dramatically increased SENP in the fatty liver and that its level was modulated by fed/fasted conditions. To define the role of hepatic SENP2 in metabolic regulation, we generated liver-specific SENP2 knockout (Senp2-LKO) mice. Senp2-LKO mice exhibited resistance to high-fat diet-induced hepatic steatosis and obesity. RNA-sequencing analysis showed that Senp2 deficiency up-regulated genes involved in fatty acid oxidation and down-regulated genes in lipogenesis in the liver. Additionally, ablation of hepatic SENP2 activated thermogenesis of adipose tissues. Improved energy homeostasis of both the liver and adipose tissues by SENP2 disruption prompted us to detect the hepatokines, with FGF21 identified as a key factor markedly elevated in Senp2-LKO mice that maintained metabolic homeostasis. Loss of FGF21 obviously reversed the positive effects of SENP2 deficiency on metabolism. Mechanistically, by screening transcriptional factors of FGF21, peroxisome proliferator-activated receptor alpha (PPARα) was defined as the mediator for SENP2 and FGF21. SENP2 interacted with PPARα and deSUMOylated it, thereby promoting ubiquitylation and subsequent degradation of PPARα, which in turn inhibited FGF21 expression and fatty acid oxidation. Consistently, SENP2 overexpression in liver facilitated development of metabolic disorders.
    CONCLUSIONS: Our finding demonstrated a key role of hepatic SENP2 in governing metabolic balance by regulating liver-adipose tissue crosstalk, linking the SUMOylation process to metabolic regulation.
  37. Nat Commun. 2021 Sep 06. 12(1): 5302
      The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.
  38. Nat Commun. 2021 Sep 10. 12(1): 5389
      Conditional overexpression of histone reader Tripartite motif containing protein 24 (TRIM24) in mouse mammary epithelia (Trim24COE) drives spontaneous development of mammary carcinosarcoma tumors, lacking ER, PR and HER2. Human carcinosarcomas or metaplastic breast cancers (MpBC) are a rare, chemorefractory subclass of triple-negative breast cancers (TNBC). Comparison of Trim24COE metaplastic carcinosarcoma morphology, TRIM24 protein levels and a derived Trim24COE gene signature reveals strong correlation with human MpBC tumors and MpBC patient-derived xenograft (PDX) models. Global and single-cell tumor profiling reveal Met as a direct oncogenic target of TRIM24, leading to aberrant PI3K/mTOR activation. Here, we find that pharmacological inhibition of these pathways in primary Trim24COE tumor cells and TRIM24-PROTAC treatment of MpBC TNBC PDX tumorspheres decreased cellular viability, suggesting potential in therapeutically targeting TRIM24 and its regulated pathways in TRIM24-expressing TNBC.