bims-proteo Biomed News
on Proteostasis
Issue of 2022‒09‒25
thirty-two papers selected by
Eric Chevet

  1. EMBO J. 2022 Sep 19. e111015
      Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold-type E3 ligase mechanism that activates the UFM1-conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.
    Keywords:  E3 ligase; enzyme substrate; post-translational modification; ribosome; ubiquitin-like modifier
  2. Comput Struct Biotechnol J. 2022 ;20 4921-4929
      Protein ubiquitination plays a vital role in controlling the degradation of intracellular proteins and in regulating cell signaling pathways. Functionally, E3 ubiquitin ligases control the transfer of ubiquitin to the target substrates. As a major family of ubiquitin E3 ligases, the structural assembly of RING E3 ligases required to exert their ubiquitin E3 ligase activity remains poorly defined. Here, we solved the crystal structure of the coiled-coil domain of TRIM75, a member of the RING E3 ligase family, which showed that two disulfide bonds stabilize two antiparallel dimers at a small crossing angle. This tetrameric conformation confers two close RING domains on the same side to form a dimer. Furthermore, this architecture allows the RING dimer to present ubiquitin to a substrate on the same side. Overall, this structure reveals a disulfide bond-mediated unique tetramer architecture and provides a tetrameric structural model through which E3 ligases exert their function.
    Keywords:  Coiled-coil domain; Crystal structure; Disulfide bond; TRIM75; Tetramerization; Ubiquitin E3 ligase
  3. Aging Cell. 2022 Sep 18. e13713
      Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age-related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub-proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age-related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein-loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst-RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst-RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst-RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.
    Keywords:  aging; autophagy; chaperones; endosomal microautophagy; exocyst complex; late endosomes; protein secretion; proteostasis
  4. EMBO Rep. 2022 Sep 20. e55099
      Stimulator of interferon genes (STING) is an essential signaling protein that is located on the endoplasmic reticulum (ER) and triggers the production of type I interferons (IFN) and proinflammatory cytokines in response to pathogenic DNA. Aberrant activation of STING is linked to autoimmune diseases. The mechanisms underlying homeostatic regulation of STING are unclear. Here, we report that UNC13D, which is associated with familial hemophagocytic lymphohistiocytosis (FHL3), is a negative regulator of the STING-mediated innate immune response. UNC13D colocalizes with STING on the ER and inhibits STING oligomerization. Cellular knockdown and knockout of UNC13D promote the production of interferon-β (IFN-β) induced by DNA viruses, but not RNA viruses. Moreover, UNC13D deficiency also increases the basal level of proinflammatory cytokines. These effects are diminished by an inhibitor of STING signaling. Furthermore, the domains involved in the UNC13D/STING interaction on both proteins are mapped. Our findings provide insight into the regulatory mechanism of STING, the previously unknown cellular function of UNC13D and the potential pathogenesis of FHL3.
    Keywords:  IFN-β; STING; UNC13D; innate immunity; proinflammatory cytokines
  5. J Biol Chem. 2022 Sep 14. pii: S0021-9258(22)00937-1. [Epub ahead of print] 102494
      Chaperones of the Hsp100/Clp family represent major components of protein homeostasis, conferring maintenance of protein activity under stress. The ClpB-type members of the family, present in bacteria, fungi, and plants, are able to resolubilize aggregated proteins. The mitochondrial member of the ClpB family in Saccharomyces cerevisiae is Hsp78. Although Hsp78 has been shown to contribute to proteostasis in elevated temperatures, the biochemical mechanisms underlying this mitochondria-specific thermotolerance are still largely unclear. To identify endogenous chaperone substrate proteins, here we generated an Hsp78-ATPase mutant with stabilized substrate binding behavior. We used two stable isotope labeling (SILAC)-based quantitative mass spectrometry approaches to analyze the role of Hsp78 during heat stress-induced mitochondrial protein aggregation and disaggregation on a proteomic level. We first identified the endogenous substrate spectrum of the Hsp78 chaperone, comprising a wide variety of proteins related to metabolic functions including energy production and protein synthesis, as well as other chaperones, indicating its crucial functions in mitochondrial stress resistance. We then compared these interaction data with aggregation and disaggregation processes in mitochondria under heat stress, which revealed specific aggregation-prone protein populations and demonstrated the direct quantitative impact of Hsp78 on stress-dependent protein solubility under different conditions. We conclude that Hsp78, together with its cofactors, represents a recovery system that protects major mitochondrial metabolic functions during heat stress as well as restores protein biogenesis capacity after the return to normal conditions.
    Keywords:  Hsp78; cell biology; chaperone; heat stress; mitochondria; protein aggregation; proteostasis; yeast
  6. Proc Natl Acad Sci U S A. 2022 Sep 27. 119(39): e2209823119
      Autophagosomes are unique organelles that form de novo as double-membrane vesicles engulfing cytosolic material for destruction. Their biogenesis involves membrane transformations of distinctly shaped intermediates whose ultrastructure is poorly understood. Here, we combine cell biology, correlative cryo-electron tomography (cryo-ET), and extensive data analysis to reveal the step-by-step structural progression of autophagosome biogenesis at high resolution directly within yeast cells. The analysis uncovers an unexpectedly thin intermembrane distance that is dilated at the phagophore rim. Mapping of individual autophagic structures onto a timeline based on geometric features reveals a dynamical change of membrane shape and curvature in growing phagophores. Moreover, our tomograms show the organelle interactome of growing autophagosomes, highlighting a polar organization of contact sites between the phagophore and organelles, such as the vacuole and the endoplasmic reticulum (ER). Collectively, these findings have important implications for the contribution of different membrane sources during autophagy and for the forces shaping and driving phagophores toward closure without a templating cargo.
    Keywords:  autophagosome biogenesis; autophagy; cryo-electron tomography; membrane structure; organelle contact sites
  7. Nature. 2022 Sep 21.
      Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.
  8. Biochim Biophys Acta Biomembr. 2022 Sep 17. pii: S0005-2736(22)00189-4. [Epub ahead of print]1864(12): 184051
      In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62 interacts with the Sec61 channel and is located on the front side of the Sec61 lateral gate, an entry site for TM domains to the lipid bilayer. Overexpression of Sec62 led to a growth defect in yeast, and we investigated its effects on protein translocation and membrane insertion by pulse labeling of Sec62 client proteins. Our data show that the insertion efficiency of marginally hydrophobic TM segments is reduced upon Sec62 overexpression. This result suggests a potential regulatory role of Sec62 as a gatekeeper of the lateral gate, thereby modulating the insertion threshold of TM segments.
    Keywords:  Biosynthesis; Marginally hydrophobic; Membrane insertion; Overexpression; Sec62; Transmembrane
  9. Autophagy. 2022 Sep 21.
      Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.
    Keywords:  ATG9; Arabidopsis; SNARE; VAMP724; VAMP726; autophagy
  10. Int Immunol. 2022 Sep 23. pii: dxac047. [Epub ahead of print]
      Polyubiquitination is a post-translational modification involved in a wide range of immunological events, including inflammatory responses, immune cell differentiation, and development of inflammatory diseases. The versatile functions of polyubiquitination are based on different types of ubiquitin linkage, which enable various UBD (ubiquitin binding domain)-containing adaptor proteins to associate and induce distinct biological outputs. A unique and atypical type of polyubiquitin chain comprising a conjugation between the N-terminal methionine of the proximal ubiquitin moiety and the C-terminal glycine of the distal ubiquitin moiety, referred to as a linear or M1-linked ubiquitin chain, has been studied exclusively within the field of immunology because it is distinct from other polyubiquitin forms: linear ubiquitin chains are generated predominantly by various inflammatory stimulants, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), and act as a critical modulator of transient and optimal signal transduction. Moreover, accumulating evidence suggests that linear ubiquitin chains are of physiological significance. Dysregulation of linear ubiquitination triggers chronic inflammation and immunodeficiency via downregulation of linear ubiquitin-dependent nuclear factor-kappa B (NF-κB) signaling and by triggering TNF-α-induced cell death, suggesting that linear ubiquitination is a homeostatic regulator of tissue-specific functions. In this review, we focus on our current understating of the molecular and cellular mechanisms by which linear ubiquitin chains control inflammatory environments. Furthermore, we review the role of linear ubiquitination on T cell development, differentiation, and function, thereby providing insight into its direct association with maintaining the immune system.
    Keywords:  Cell death; Inflammation; LUBAC; T cell; TNF signaling
  11. Nat Metab. 2022 Sep;4(9): 1166-1184
      Adipose tissue undergoes thermogenic remodeling in response to thermal stress and metabolic cues, playing a crucial role in regulating energy expenditure and metabolic homeostasis. Endoplasmic reticulum (ER) stress is associated with adipose dysfunction in obesity and metabolic disease. It remains unclear, however, if ER stress-signaling in adipocytes mechanistically mediates dysregulation of thermogenic fat. Here we show that inositol-requiring enzyme 1α (IRE1α), a key ER stress sensor and signal transducer, acts in both white and beige adipocytes to impede beige fat activation. Ablation of adipocyte IRE1α promotes browning/beiging of subcutaneous white adipose tissue following cold exposure or β3-adrenergic stimulation. Loss of IRE1α alleviates diet-induced obesity and augments the anti-obesity effect of pharmacologic β3-adrenergic stimulation. Notably, IRE1α suppresses stimulated lipolysis and degrades Ppargc1a messenger RNA through its RNase activity to downregulate the thermogenic gene program. Hence, blocking IRE1α bears therapeutic potential in unlocking adipocytes' thermogenic capacity to combat obesity and metabolic disorders.
  12. Nat Commun. 2022 Sep 22. 13(1): 5565
      Microtubules play a crucial role during the establishment and maintenance of cell polarity. In fission yeast cells, the microtubule plus-end tracking proteins (+TIPs) (including the CLIP-170 homologue Tip1) regulate microtubule dynamics and also transport polarity factors to the cell cortex. Here, we show that the E3 ubiquitin ligase Dma1 plays an unexpected role in controlling polarized growth through ubiquitinating Tip1. Dma1 colocalizes with Tip1 to cortical sites at cell ends, and is required for ubiquitination of Tip1. Although the absence of dma1+ does not cause apparent polar growth defects in vegetatively growing cells, Dma1-mediated Tip1 ubiquitination is required to restrain polar growth upon DNA replication stress. This mechanism is distinct from the previously recognized calcineurin-dependent inhibition of polarized growth. In this work, we establish a link between Dma1-mediated Tip1 ubiquitination and DNA replication or DNA damage checkpoint-dependent inhibition of polarized growth in fission yeast.
  13. J Cell Biol. 2022 Nov 07. pii: e202109137. [Epub ahead of print]221(11):
      Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.
  14. Cancer Cell. 2022 Aug 29. pii: S1535-6108(22)00380-4. [Epub ahead of print]
      Activation of unfolded protein responses (UPRs) in cancer cells undergoing endoplasmic reticulum (ER) stress promotes survival. However, how UPR in tumor cells impacts anti-tumor immune responses remains poorly described. Here, we investigate the role of the UPR mediator pancreatic ER kinase (PKR)-like ER kinase (PERK) in cancer cells in the modulation of anti-tumor immunity. Deletion of PERK in cancer cells or pharmacological inhibition of PERK in melanoma-bearing mice incites robust activation of anti-tumor T cell immunity and attenuates tumor growth. PERK elimination in ER-stressed malignant cells triggers SEC61β-induced paraptosis, thereby promoting immunogenic cell death (ICD) and systemic anti-tumor responses. ICD induction in PERK-ablated tumors stimulates type I interferon production in dendritic cells (DCs), which primes CCR2-dependent tumor trafficking of common-monocytic precursors and their intra-tumor commitment into monocytic-lineage inflammatory Ly6C+CD103+ DCs. These findings identify how tumor cell-derived PERK promotes immune evasion and highlight the potential of PERK-targeting therapies in cancer immunotherapy.
    Keywords:  PERK; immunogenic cell death; tumor immunity; type I IFN; unfolded protein responses
  15. Nat Biotechnol. 2022 Sep 22.
      Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.
  16. Autophagy. 2022 Sep 21. 1-16
      How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.
    Keywords:  3-MA; Con A; MG115; calpeptin; proteasome; rapamycin
  17. Front Cell Dev Biol. 2022 ;10 961675
      Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.
    Keywords:  Ufl1; Ufl1f/f KRT14Cre/+; Ufmylation modification; endothelin-1; pigmentation
  18. Cancer Res. 2022 Sep 20. pii: CAN-22-1744. [Epub ahead of print]
      Protein synthesis supports robust immune responses. Nutrient competition and global cell stressors in the tumor microenvironment (TME) may impact protein translation in T cells and antitumor immunity. Using human and mouse tumors, we demonstrated here that protein translation in T cells is repressed in solid tumors. Reduced glucose availability to T cells in the TME led to activation of the unfolded protein response (UPR) element eIF2a. Genetic mouse models revealed that translation attenuation mediated by activated p-eIF2a undermines the ability of T cells to suppress tumor growth. Reprogramming T cell metabolism was able to alleviate p-eIF2a accumulation and translational attenuation in the TME, allowing for sustained protein translation. Metabolic and pharmacological approaches showed that proteasome activity mitigates induction of p-eIF2a to support optimal antitumor T cell function, protecting from translation attenuation and enabling prolonged cytokine synthesis in solid tumors. Together, these data identify a new therapeutic avenue to fuel the efficacy of tumor immunotherapy.
  19. Cell Rep. 2022 Sep 20. pii: S2211-1247(22)01233-5. [Epub ahead of print]40(12): 111396
      Deubiquitinating enzymes are key regulators of the ubiquitin-proteasome system and cell cycle, and their dysfunction leads to tumorigenesis. Our in vivo drop-out screens in patient-derived xenograft models identify USP7 as a regulator of melanoma. We show that USP7 downregulation induces cellular senescence, arresting melanoma growth in vivo and proliferation in vitro in BRAF- and NRAS-mutant melanoma. We provide a comprehensive understanding of targets and networks affected by USP7 depletion by performing a global transcriptomic and proteomics analysis. We show that RRM2 is a USP7 target and is regulated by USP7 during S phase of the cell cycle. Ectopic expression of RRM2 in USP7-depleted cells rescues the senescent phenotype. Pharmacological inhibition of USP7 by P5091 phenocopies the shUSP7-induced senescent phenotype. We show that the bifunctional histone deacetylase (HDAC)/LSD1 inhibitor domatinostat has an additive antitumor effect, eliminating P5091-induced senescent cells, paving the way to a therapeutic combination for individuals with melanoma.
    Keywords:  CP: Cancer; DUBs; PDX; cellular senescence; combination therapy; metastatic melanoma
  20. STAR Protoc. 2022 Sep 21. pii: S2666-1667(22)00590-1. [Epub ahead of print]3(4): 101710
      Mitochondrial polymerase gamma (PolγA) is the only replicative polymerase in mitochondria. To determine PolγA ubiquitylation in cells, Flag-PolγA and MITOL are overexpressed, and subsequently the immunoprecipitated Flag-PolγA is checked for ubiquitylation. Alternately, in vitro synthesized PolγA and MITOL are used to determine whether PolγA is ubiquitylated. Either anti-ubiquitin or anti-Flag antibody is used to detect the ubiquitylated product. Thus, we provide a detailed, reliable, highly reproducible protocol for detecting ubiquitylation of PolγA by MITOL, both in cells and in vitro. For complete details on the use and execution of this protocol, please refer to Hussain et al. (2021).
    Keywords:  Cell biology; Genetics; Molecular biology; Protein biochemistry; Protein expression and purification
  21. Trends Cell Biol. 2022 Sep 20. pii: S0962-8924(22)00208-2. [Epub ahead of print]
      Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.
    Keywords:  aggrephagy; autophagy; autophagy receptor; biocondensophagy; biomolecular condensate; phase separation; ubiquitin
  22. PLoS Genet. 2022 Sep 23. 18(9): e1010425
      Transcriptional elongation is a universal and critical step during gene expression. The super elongation complex (SEC) regulates the rapid transcriptional induction by mobilizing paused RNA polymerase II (Pol II). Dysregulation of SEC is closely associated with human diseases. However, the physiological role of SEC during development and homeostasis remains largely unexplored. Here we studied the function of SEC in adipogenesis by manipulating an essential scaffold protein AF4/FMR2 family member 4 (AFF4), which assembles and stabilizes SEC. Knockdown of AFF4 in human mesenchymal stem cells (hMSCs) and mouse 3T3-L1 preadipocytes inhibits cellular adipogenic differentiation. Overexpression of AFF4 enhances adipogenesis and ectopic adipose tissue formation. We further generate Fabp4-cre driven adipose-specific Aff4 knockout mice and find that AFF4 deficiency impedes adipocyte development and white fat depot formation. Mechanistically, we discover AFF4 regulates autophagy during adipogenesis. AFF4 directly binds to autophagy-related protein ATG5 and ATG16L1, and promotes their transcription. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overepressing cells, while overexpression of ATG5 and ATG16L1 rescues the impaired adipogenesis in Aff4-knockout cells. Collectively, our results unveil the functional importance of AFF4 in regulating autophagy and adipogenic differentiation, which broaden our understanding of the transcriptional regulation of adipogenesis.
  23. Acta Neuropathol. 2022 Sep 19.
      ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset.
  24. Nat Commun. 2022 Sep 20. 13(1): 5495
      Conditional degron tags (CDTs) are a powerful tool for target validation that combines the kinetics and reversible action of pharmacological agents with the generalizability of genetic manipulation. However, successful design of a CDT fusion protein often requires a prolonged, ad hoc cycle of construct design, failure, and re-design. To address this limitation, we report here a system to rapidly compare the activity of five unique CDTs: AID/AID2, IKZF3d, dTAG, HaloTag, and SMASh. We demonstrate the utility of this system against 16 unique protein targets. We find that expression and degradation are highly dependent on the specific CDT, the construct design, and the target. None of the CDTs leads to efficient expression and/or degradation across all targets; however, our systematic approach enables the identification of at least one optimal CDT fusion for each target. To enable the adoption of CDT strategies more broadly, we have made these reagents, and a detailed protocol, available as a community resource.
  25. Biochim Biophys Acta Biomembr. 2022 Sep 16. pii: S0005-2736(22)00188-2. [Epub ahead of print]1864(12): 184050
      Most eukaryotic secretory and membrane proteins are funneled by the Sec61 complex into the secretory pathway. Furthermore, some substrate peptides rely on two essential accessory proteins, Sec62 and Sec63, being present to assist with their translocation via the Sec61 channel in post-translational translocation. Cryo-electron microscopy (cryo-EM) recently succeeded in determining atomistic structures of unbound and signal sequence-engaged Sec complexes from Saccharomyces cerevisiae, involving the Sec61 channel and the proteins Sec62, Sec63, Sec71 and Sec72. In this study, we investigated the conformational effects of Sec62 on Sec61. Indeed, we observed in molecular dynamics simulations that the conformational dynamics of lateral gate, plug and pore region of Sec61 are altered by the presence/absence of Sec62. In molecular dynamics simulations that were started from the cryo-EM structures of Sec61 coordinated to Sec62 or of apo Sec61, we observed that the luminal side of the lateral gate gradually adopts a closed conformation similar to the apo state during unbound state simulations. In contrast, it adopts a wider conformation in the bound state. Furthermore, we demonstrate that the conformation of the active (substrate-bound) state of the Sec61 channel shifts toward an alternative conformation in the absence of the substrate. We suggest that the signal peptide holds/stabilizes the active state conformation of Sec61 during post-translational translocation. Thus, our study explains the effect of Sec62 on the conformation of the Sec61 channel and describes the conformational transitions of Sec61 channel.
    Keywords:  Accessory protein; Molecular dynamics simulation; Protein translocation
  26. Sci Adv. 2022 Sep 23. 8(38): eabq8303
      Membrane thinning by rhomboid proteins has been proposed to reduce hydrophobic mismatch, providing a unique environment for important functions ranging from intramembrane proteolysis to retrotranslocation in protein degradation. We show by in vitro reconstitution and solid-state nuclear magnetic resonance that the lipid environment of the Escherichia coli rhomboid protease GlpG influences its activity with an optimal hydrophobic membrane thickness between 24 and 26 Å. While phosphatidylcholine membranes are only negligibly altered by GlpG, in an E. coli-relevant lipid mix of phosphatidylethanolamine and phosphatidylglycerol, a thinning by 1.1 Å per leaflet is observed. Protease activity is strongly correlated with membrane thickness and shows no lipid headgroup specificity. We infer from these results that, by adjusting the thickness of specific membrane domains, membrane proteins shape the bilayer for their specific needs.
  27. RSC Chem Biol. 2022 Aug 31. 3(9): 1144-1153
      Dysregulated transcription factors (TFs) that rewire gene expression circuitry are frequently identified as key players in disease. Although several TFs have been drugged with small molecules, the majority of oncogenic TFs are not currently pharmaceutically tractable due to their paucity of ligandable pockets. The first generation of transcription factor targeting chimeras (TRAFTACs) was developed to target TFs for proteasomal degradation by exploiting their DNA binding ability. In the current study, we have developed the second generation TRAFTACs ("oligoTRAFTACs") composed of a TF-binding oligonucleotide and an E3 ligase-recruiting ligand. Herein, we demonstrate the development of oligoTRAFTACs to induce the degradation of two oncogenic TFs, c-Myc and brachyury. In addition, we show that brachyury can be successfully degraded by oligoTRAFTACs in chordoma cell lines. Furthermore, zebrafish experiments demonstrate in vivo oligoTRAFTAC activity. Overall, our data demonstrate oligoTRAFTACs as a generalizable platform towards difficult-to-drug TFs and their degradability via the proteasomal pathway.
  28. Nat Commun. 2022 Sep 19. 13(1): 5491
      Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.
  29. Cell Mol Life Sci. 2022 Sep 22. 79(10): 526
      CAPRIN1 is a ubiquitously expressed protein, abundant in the brain, where it regulates the transport and translation of mRNAs of genes involved in synaptic plasticity. Here we describe two unrelated children, who developed early-onset ataxia, dysarthria, cognitive decline and muscle weakness. Trio exome sequencing unraveled the identical de novo c.1535C > T (p.Pro512Leu) missense variant in CAPRIN1, affecting a highly conserved residue. In silico analyses predict an increased aggregation propensity of the mutated protein. Indeed, overexpressed CAPRIN1P512L forms insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders (ATXN2, GEMIN5, SNRNP200 and SNCA). Moreover, the CAPRIN1P512L mutation in isogenic iPSC-derived cortical neurons causes reduced neuronal activity and altered stress granule dynamics. Furthermore, nano-differential scanning fluorimetry reveals that CAPRIN1P512L aggregation is strongly enhanced by RNA in vitro. These findings associate the gain-of-function Pro512Leu mutation to early-onset ataxia and neurodegeneration, unveiling a critical residue of CAPRIN1 and a key role of RNA-protein interactions.
    Keywords:  CRISPR/Cas9; De novo variant; Neurodegeneration; Prion-like domain; Protein misfolding
  30. Mol Cell Proteomics. 2022 Sep 14. pii: S1535-9476(22)00221-3. [Epub ahead of print] 100413
      The assembly of proteins and peptides into amyloid fibrils is causally linked to serious disorders such as Alzheimer's Disease. Multiple proteins have been shown to prevent amyloid formation in vitro and in vivo, ranging from highly specific chaperone-client pairs to completely non-specific binding of aggregation-prone peptides. The underlying interactions remain elusive. Here, we turn to the machine learning-based structure prediction algorithm AlphaFold2 (AF2) to obtain models for the non-specific interactions of β-lactoglobulin (βLG), transthyretin (TTR), or Thioredoxin 80 (T80) with the model amyloid peptide Amyloid β (Aβ), and the highly specific complex between the BRICHOS chaperone domain of lung surfactant protein C (CTC) and its polyvaline target. Using a combination of native mass spectrometry (MS) and ion mobility MS, we show that non-specific chaperoning is driven predominantly by hydrophobic interactions of Aβ with hydrophobic surfaces in βLG, TTR, and T80, and in part regulated by oligomer stability. For CTC, native MS and hydrogen-deuterium exchange MS reveal that a disordered region recognizes the polyvaline target by forming a complementary β-strand. Hence, we show that AF2 and MS can yield atomistic models of hard-to-capture protein interactions that reveal different chaperoning mechanisms based on separate ligand properties and may provide possible clues for specific therapeutic intervention.
    Keywords:  Structural Proteomics; machine learning; molecular chaperones; protein misfolding
  31. Elife. 2022 Sep 23. pii: e79771. [Epub ahead of print]11
      Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.
    Keywords:  biochemistry; cell biology; chemical biology; human
  32. EMBO J. 2022 Sep 19. e112349
      Cells are able to adapt their growth to external mechanical strain. A recent study by Phuyal et al (2022) has shown that these responses depend on the heterodimerization of two small GTPases.