bims-proteo Biomed News
on Proteostasis
Issue of 2022‒09‒11
thirty-two papers selected by
Eric Chevet

  1. Cell Chem Biol. 2022 Aug 30. pii: S2451-9456(22)00312-9. [Epub ahead of print]
      Proteolysis-targeting chimeras (PROTACs) bring a protein of interest (POI) into spatial proximity of an E3 ubiquitin ligase, promoting POI ubiquitylation and proteasomal degradation. PROTACs rely on endogenous cellular machinery to mediate POI degradation, therefore the subcellular location of the POI and access to the E3 ligase being recruited potentially impacts PROTAC efficacy. To interrogate whether the subcellular context of the POI influences PROTAC-mediated degradation, we expressed either Halo or FKBP12F36V (dTAG) constructs consisting of varying localization signals and tested the efficacy of their degradation by von Hippel-Lindau (VHL)- or cereblon (CRBN)-recruiting PROTACs targeting either Halo or dTAG. POIs were localized to the nucleus, cytoplasm, outer mitochondrial membrane, endoplasmic reticulum, Golgi, peroxisome or lysosome. Differentially localized Halo or FKBP12F36V proteins displayed varying levels of degradation using the same respective PROTACs, suggesting therefore that the subcellular context of the POI can influence the efficacy of PROTAC-mediated POI degradation.
    Keywords:  CRBN; E3; Halo; HiBiT; VHL; cereblon; dTAG; degrader; protein localization; proteolysis-targeting chimera (PROTAC); targeted protein degradation (TPD)
  2. Cell Chem Biol. 2022 Aug 29. pii: S2451-9456(22)00311-7. [Epub ahead of print]
      Targeted protein degradation (TPD) uses small molecules to recruit E3 ubiquitin ligases into the proximity of proteins of interest, inducing ubiquitination-dependent degradation. A major bottleneck in the TPD field is the lack of accessible E3 ligase ligands for developing degraders. To expand the E3 ligase toolbox, we sought to convert the Kelch-like ECH-associated protein 1 (KEAP1) inhibitor KI696 into a recruitment handle for several targets. While we were able to generate KEAP1-recruiting degraders of BET family and murine focal adhesion kinase (FAK), we discovered that the target scope of KEAP1 was narrow, as targets easily degraded using a cereblon (CRBN)-recruiting degrader were refractory to KEAP1-mediated degradation. Linking the KEAP1-binding ligand to a CRBN-binding ligand resulted in a molecule that induced degradation of KEAP1 but not CRBN. In sum, we characterize tool compounds to explore KEAP1-mediated ubiquitination and delineate the challenges of exploiting new E3 ligases for generating bivalent degraders.
    Keywords:  BRD4; FAK; KEAP1; PROTACs; degrader; targeted protein degradation
  3. Front Cell Dev Biol. 2022 ;10 915065
      The Bcl-2 family proteins BAK and BAX control the crucial step of pore formation in the mitochondrial outer membrane during intrinsic apoptosis. Bcl-2-related ovarian killer (BOK) is a Bcl-2 family protein with a high sequence similarity to BAK and BAX. However, intrinsic apoptosis can proceed in the absence of BOK. Unlike BAK and BAX, BOK is primarily located on the endoplasmic reticulum (ER) and Golgi membranes, suggesting a role for BOK in regulating ER homeostasis. In this study, we report that BOK is required for a full ER stress response. Employing previously characterized fluorescent protein-based ER stress reporter cell systems, we show that BOK-deficient cells have an attenuated response to ER stress in all three signaling branches of the unfolded protein response. Fluo-4-based confocal Ca2+ imaging revealed that disruption of ER proteostasis in BOK-deficient cells was not linked to altered ER Ca2+ levels. Fluorescence recovery after photobleaching (FRAP) experiments using GRP78/BiP-eGFP demonstrated that GRP78 motility was significantly lower in BOK-deficient cells. This implied that less intraluminal GRP78 was freely available and more of the ER chaperone bound to unfolded proteins. Collectively, these experiments suggest a new role for BOK in the protection of ER proteostasis and cellular responses to ER stress.
    Keywords:  Bcl-2 family; ER stress; ER stress reporters; calcium signaling; live-cell imaging; proteostasis; unfolded protein response
  4. Neurochem Res. 2022 Sep 08.
      The endoplasmic reticulum (ER) is the primary site of intracellular quality control involved in the recognition and degradation of unfolded proteins. A variety of stresses, including hypoxia and glucose starvation, can lead to accumulation of unfolded proteins triggering the ER-associated degradation (ERAD) pathway. Suppressor Enhancer Lin12/Notch1 Like (Sel1l) acts as a "gate keeper" in the quality control of de novo synthesized proteins and complexes with the ubiquitin ligase Hrd1 in the ER membrane. We previously demonstrated that ER stress-induced aberrant neural stem cell (NSC) differentiation and inhibited neurite outgrowth. Inhibition of neurite outgrowth was associated with increased Hrd1 expression; however, the contribution of Sel1l remained unclear. To investigate whether ER stress is induced during normal neuronal differentiation, we semi-quantitatively evaluated mRNA expression levels of unfolded protein response (UPR)-related genes in P19 embryonic carcinoma cells undergoing neuronal differentiation in vitro. Stimulation with all-trans retinoic acid (ATRA) for 4 days induced the upregulation of Nestin and several UPR-related genes (Atf6, Xbp1, Chop, Hrd1, and Sel1l), whereas Atf4 and Grp78/Bip were unchanged. Small-interfering RNA (siRNA)-mediated knockdown of Sel1l uncovered that mRNA levels of the neural progenitor marker Math1 (also known as Atoh1) and the neuronal marker Math3 (also known as Atoh3 and NeuroD4) were significantly suppressed at 4 days after ATRA stimulation. Consistent with this result, Sel1l silencing significantly reduced protein levels of immature neuronal marker βIII-tubulin (also known as Tuj-1) at 8 days after induction of neuronal differentiation, whereas synaptogenic factors, such as cell adhesion molecule 1 (CADM1) and SH3 and multiple ankyrin repeat domain protein 3 (Shank3) were accumulated in Sel1l silenced cells. These results indicate that neuronal differentiation triggers ER stress and suggest that Sel1l may facilitate neuronal lineage through the regulation of Math1 and Math3 expression.
    Keywords:  Endoplasmic reticulum-associated degradation (ERAD); Hrd1; Neuronal differentiation; Sel1l; Unfolded protein response (UPR)
  5. Nat Cell Biol. 2022 Sep 08.
      While acetylated, RNA-binding-deficient TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) comprised of TDP-43-containing liquid outer shells and liquid centres of HSP70-family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we show that transient oxidative stress, proteasome inhibition or inhibition of the ATP-dependent chaperone activity of HSP70 provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independently of RNA binding or stress granules. Isotope labelling mass spectrometry was used to identify that phase-separated cytoplasmic TDP-43 is bound by the small heat-shock protein HSPB1. Binding is direct, mediated through TDP-43's RNA binding and low-complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced TDP-43 droplets. A decrease in HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion was identified in spinal motor neurons of patients with ALS containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.
  6. Cell Host Microbe. 2022 Sep 01. pii: S1931-3128(22)00407-3. [Epub ahead of print]
      Chlamydia trachomatis is the leading cause of sexually transmitted bacterial infections and a major threat to women's reproductive health in particular. This obligate intracellular pathogen resides and replicates within a cellular compartment termed an inclusion, where it is sheltered by unknown mechanisms from gamma-interferon (IFNγ)-induced cell-autonomous host immunity. Through a genetic screen, we uncovered the Chlamydia inclusion membrane protein gamma resistance determinant (GarD) as a bacterial factor protecting inclusions from cell-autonomous immunity. In IFNγ-primed human cells, inclusions formed by garD loss-of-function mutants become decorated with linear ubiquitin and are eliminated. Leveraging cellular genome-wide association data, we identified the ubiquitin E3 ligase RNF213 as a candidate anti-Chlamydia protein. We demonstrate that IFNγ-inducible RNF213 facilitates the ubiquitylation and destruction of GarD-deficient inclusions. Furthermore, we show that GarD operates as a cis-acting stealth factor barring RNF213 from targeting inclusions, thus functionally defining GarD as an RNF213 antagonist essential for chlamydial growth during IFNγ-stimulated immunity.
    Keywords:  Chlamydia; NDP52; RNF213; TAX1BP1; autophagy; cell-autonomous immunity; interferon; interferon-stimulated genes; optineurin; ubiquitylation
  7. Mol Biol Cell. 2022 Sep 08. mbcE22060233
      RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12 that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure-activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12 that has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promotes proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.
  8. Methods Mol Biol. 2022 ;2543 71-82
      In conditions of cellular stress and nutrient shortage, macroautophagy (hereafter referred to as autophagy) assures the degradation of dysfunctional macromolecules and organelles as it liberates energy resources via the breakdown of dispensable cellular components. Morphologically, autophagy is characterized by the formation of double-membraned autophagosomes that facilitate the isolation of autophagic cargo for subsequent lysosomal degradation at low pH. Sequestosome-1 (SQSTM1, better known as ubiquitin-binding protein p62), is an autophagosomal cargo receptor that targets proteins for selective autophagic degradation. Indeed, the redistribution of tandem mCherry and enhanced green fluorescent protein (mCherry-EGFP)-conjugated p62 from the cytosol into nascent autophagosomes constitutes a phenotype applicable to microscopic analysis. Furthermore, the differential pH sensitivity of mCherry and EGFP allows the visualization of autophagic flux due to the selective decrease of the EGFP signal upon fusion of autophagosomes with lysosomes. Here, we describe a method employing automated confocal cellular imaging for the study of autophagic degradation that is amenable to systems biology approaches.
    Keywords:  Autophagic flux; Autophagy; Image analysis; Lysosomal degradation
  9. Redox Biol. 2022 Aug 27. pii: S2213-2317(22)00227-0. [Epub ahead of print]56 102455
      N-glycosylation and disulfide bond formation are two essential steps in protein folding that occur in the endoplasmic reticulum (ER) and reciprocally influence each other. Here, to analyze crosstalk between N-glycosylation and oxidation, we investigated how the protein disulfide oxidase ERO1-alpha affects glycosylation of the angiogenic VEGF121, a key regulator of vascular homeostasis. ERO1 deficiency, while retarding disulfide bond formation in VEGF121, increased utilization of its single N-glycosylation sequon, which lies close to an intra-polypeptide disulfide bridge, and concomitantly slowed its secretion. Unbiased mass-spectrometric analysis revealed interactions between VEGF121 and N-glycosylation pathway proteins in ERO1-knockout (KO), but not wild-type cells. Notably, MAGT1, a thioredoxin-containing component of the post-translational oligosaccharyltransferase complex, was a major hit exclusive to ERO1-deficient cells. Thus, both a reduced rate of formation of disulfide bridges, and the increased trapping potential of MAGT1 may increase N-glycosylation of VEGF121. Extending our investigation to tissues, we observed altered lectin staining of ERO1 KO breast tumor xenografts, implicating ERO1 as a physiologic regulator of protein N-glycosylation. Our study, highlighting the effect of ERO1 loss on N-glycosylation of proteins, is particularly relevant not only to angiogenesis but also to other cancer patho-mechanisms in light of recent findings suggesting a close causal link between alterations in protein glycosylation and cancer development.
    Keywords:  Angiogenesis; ERO1 alpha; N-glycosylation; Oxidative folding; VEGFA
  10. Nature. 2022 Sep 07.
      Lysosomal dysfunction has been increasingly linked to disease and normal ageing1,2. Lysosomal membrane permeabilization (LMP), a hallmark of lysosome-related diseases, can be triggered by diverse cellular stressors3. Given the damaging contents of lysosomes, LMP must be rapidly resolved, although the underlying mechanisms are poorly understood. Here, using an unbiased proteomic approach, we show that LMP stimulates a phosphoinositide-initiated membrane tethering and lipid transport (PITT) pathway for rapid lysosomal repair. Upon LMP, phosphatidylinositol-4 kinase type 2α (PI4K2A) accumulates rapidly on damaged lysosomes, generating high levels of the lipid messenger phosphatidylinositol-4-phosphate. Lysosomal phosphatidylinositol-4-phosphate in turn recruits multiple oxysterol-binding protein (OSBP)-related protein (ORP) family members, including ORP9, ORP10, ORP11 and OSBP, to orchestrate extensive new membrane contact sites between damaged lysosomes and the endoplasmic reticulum. The ORPs subsequently catalyse robust endoplasmic reticulum-to-lysosome transfer of phosphatidylserine and cholesterol to support rapid lysosomal repair. Finally, the lipid transfer protein ATG2 is also recruited to damaged lysosomes where its activity is potently stimulated by phosphatidylserine. Independent of macroautophagy, ATG2 mediates rapid membrane repair through direct lysosomal lipid transfer. Together, our findings identify that the PITT pathway maintains lysosomal membrane integrity, with important implications for numerous age-related diseases characterized by impaired lysosomal function.
  11. EMBO J. 2022 Sep 05. e112384
      Complex metabolic diseases such as diabetes and non-alcoholic fatty liver disease have been associated with aberrant lipid metabolism and lipotoxicity. To maintain lipid homeostasis and escape lipotoxicity, cells deploy a plethora of mechanisms, the most fascinating of which relying on a sense-and-response circuit. New work by Volkmar et al reveals an auto-regulated pathway formed by a lipid hydrolase and a lipid-sensitive E3 ubiquitin ligase playing hide-and-seek to warrant membrane function in stressed cells.
  12. Methods Mol Biol. 2022 ;2543 155-166
      Autophagy and ER stress are most often studied employing a Western blotting approach to the measurement of autophagy by LC3B upregulation and the ER stress sensor signaling proteins PERK (protein kinase R-like endoplasmic reticulum kinase), IRE1, and ATF6 which initiate protein refolding and elongation of the ER until ER homeostasis is returned. If the misfolding of proteins is increased, then ER stress is maintained, and microautophagy of the ER or specifically reticulophagy occurs. However, LC3B, PERK, protein misfolding, and changes in ER mass (reticulophagy) can also be measured in a cell cycle-dependent manner by flow cytometry and the use of antibodies, protein misfolding, and ER tracking fluorescent probes.
    Keywords:  Autophagy; ER stress; Misfolded proteins; PERK; Reticulophagy
  13. Oncogene. 2022 Sep 06.
      Most of the organs of the digestive tract comprise secretory epithelia that require specialized molecular machines to achieve their functions. As such anterior gradient (AGR) proteins, which comprise AGR1, AGR2, and AGR3, belong to the protein disulfide isomerase family, and are involved in secretory and transmembrane protein biogenesis in the endoplasmic reticulum. They are generally expressed in epithelial cells with high levels in most of the digestive tract epithelia. To date, the vast majority of the reports concern AGR2, which has been shown to exhibit various subcellular localizations and exert pro-oncogenic functions. AGR2 overexpression has recently been associated with a poor prognosis in digestive cancers. AGR2 is also involved in epithelial homeostasis. Its deletion in mice results in severe diffuse gut inflammation, whereas in inflammatory bowel diseases, the secretion of AGR2 in the extracellular milieu participates in the reshaping of the cellular microenvironment. AGR2 thus plays a key role in inflammation and oncogenesis and may represent a therapeutic target of interest. In this review, we summarize the already known roles and mechanisms of action of the AGR family proteins in digestive diseases, their expression in the healthy digestive tract, and in digestive oncology. At last, we discuss the potential diagnostic and therapeutic implications underlying the biology of AGR proteins.
  14. Science. 2022 Sep 08. eabn5637
      Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Here, through genetic screens in defined nutrient conditions we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable owing to a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.
  15. J Cell Biol. 2022 Oct 03. pii: e202202149. [Epub ahead of print]221(10):
      The chaperone-mediated sequestration of misfolded proteins into inclusions is a pivotal cellular strategy to maintain proteostasis in Saccharomyces cerevisiae, executed by small heat shock proteins (sHsps) Hsp42 and Btn2. Direct homologs of Hsp42 and Btn2 are absent in other organisms, questioning whether sequestration represents a conserved proteostasis strategy and, if so, which factors are involved. We examined sHsps from Escherchia coli, Caenorhabditis elegans, and humans for their ability to complement the defects of yeast sequestrase mutants. We show that sequestration of misfolded proteins is an original and widespread activity among sHsps executed by specific family members. Sequestrase positive C. elegans' sHsps harbor specific sequence features, including a high content of aromatic and methionine residues in disordered N-terminal extensions. Those sHsps buffer limitations in Hsp70 capacity in C. elegans WT animals and are upregulated in long-lived daf-2 mutants, contributing to lifespan extension. Cellular protection by sequestration of misfolded proteins is, therefore, an evolutionarily conserved activity of the sHsp family.
  16. Annu Rev Microbiol. 2022 Sep 08. 76 211-233
      Ubiquitination is a posttranslational modification that regulates a multitude of cellular functions. Pathogens, such as bacteria and viruses, have evolved sophisticated mechanisms that evade or counteract ubiquitin-dependent host responses, or even exploit the ubiquitin system to their own advantage. This is largely done by numerous pathogen virulence factors that encode E3 ligases and deubiquitinases, which are often used as weapons in pathogen-host cell interactions. Moreover, upon pathogen attack, host cellular signaling networks undergo major ubiquitin-dependent changes to protect the host cell, including coordination of innate immunity, remodeling of cellular organelles, reorganization of the cytoskeleton, and reprogramming of metabolic pathways to restrict growth of the pathogen. Here we provide mechanistic insights into ubiquitin regulation of host-pathogen interactions and how it affects bacterial and viral pathogenesis and the organization and response of the host cell.
    Keywords:  bacterial effectors; host-pathogen interactions; organelle remodeling; ubiquitin
  17. J Biol Chem. 2022 Sep 05. pii: S0021-9258(22)00907-3. [Epub ahead of print] 102464
      Apoptosis inducing factor (AIF) is a mitochondrion-localized flavoprotein with NADH oxidase activity. AIF normally acts as an oxidoreductase to catalyze the transfer of electrons between molecules, but it can also kill cells when exposed to certain stimuli. For example, intact AIF is cleaved upon exposure to DNA-damaging agents such as etoposide, and truncated AIF (tAIF) is released from the mitochondria to the cytoplasm and translocated to the nucleus where it induces apoptosis. Although the serial events during tAIF-mediated apoptosis and the transition of AIF function have been widely studied from various perspectives, their underlying regulatory mechanisms and the factors involved are not fully understood. Here, we demonstrated that tAIF is a target of the covalent conjugation of the ubiquitin-like moiety ISG15 (referred to as ISGylation), which is mediated by the ISG15 E3 ligase HERC5. In addition, ISGylation increases the stability of tAIF protein as well as its K6-linked polyubiquitination. Moreover, we found that ISGylation increases the nuclear translocation of tAIF upon cytotoxic etoposide treatment, subsequently causing apoptotic cell death in human lung A549 carcinoma cells. Collectively, these results suggest that HERC5-mediated ISG15 conjugation is a key factor in the positive regulation of tAIF-mediated apoptosis, highlighting a novel role of post-translational ISG15 modification as a switch that allows cells to live or die under the stress that triggers tAIF release.
    Keywords:  A549; AIF; Apoptosis; Etoposide; HERC5; ISG15; Ubiquitin-like
  18. J Biol Chem. 2022 Sep 02. pii: S0021-9258(22)00900-0. [Epub ahead of print] 102457
      AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single-molecule dwell time using TIRF (Total Internal Reflection Fluorescence) microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and composition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted substrate release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high-complexity motif containing polar and charged residues also promoted release. We further investigated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase-protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. Although polyG and GAr are both inhibitory for unfolding, they act in different ways. GAr motifs only negatively affected degradation of highly stable substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate characteristics that affect unfolding and degradation by ClpXP.
    Keywords:  ATP-dependent protease; enzyme kinetics; protein degradation; protein sequence; single-molecule biophysics
  19. Oncogene. 2022 Sep 06.
      Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.
  20. Sci Adv. 2022 Sep 09. 8(36): eabn0756
      Evolutionary profiling has been largely limited to the nucleotide level. Using consistent proteomic methods, we quantified proteomic and phosphoproteomic layers in fibroblasts from 11 common mammalian species, with transcriptomes as reference. Covariation analysis indicates that transcript and protein expression levels and variabilities across mammals remarkably follow functional role, with extracellular matrix-associated expression being the most variable, demonstrating strong transcriptome-proteome coevolution. The biological variability of gene expression is universal at both interindividual and interspecies scales but to a different extent. RNA metabolic processes particularly show higher interspecies versus interindividual variation. Our results further indicate that while the ubiquitin-proteasome system is strongly conserved in mammals, lysosome-mediated protein degradation exhibits remarkable variation between mammalian lineages. In addition, the phosphosite profiles reveal a phosphorylation coevolution network independent of protein abundance.
  21. Biochem Biophys Res Commun. 2022 Aug 27. pii: S0006-291X(22)01179-2. [Epub ahead of print]628 68-75
      PROTACs have emerged as a new class of drugs that can target the "undruggable" proteome by hijacking the ubiquitin proteasome system. Despite PROTACs' success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using "TUBEs" as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed "UbMax" - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.
    Keywords:  Drug discovery; PROTACs; TUBE technology; Targeted protein degradation; Ubiquitination
  22. Aging Cell. 2022 Sep 10. e13707
      Senescent cells accumulate in tissues over time, favoring the onset and progression of multiple age-related diseases. Senescent cells present a remarkable increase in lysosomal mass and elevated autophagic activity. Here, we report that two main autophagic pathways macroautophagy (MA) and chaperone-mediated autophagy (CMA) are constitutively upregulated in senescent cells. Proteomic analyses of the subpopulations of lysosomes preferentially engaged in each of these types of autophagy revealed profound quantitative and qualitative changes in senescent cells, affecting both lysosomal resident proteins and cargo proteins delivered to lysosomes for degradation. These studies have led us to identify resident lysosomal proteins that are highly augmented in senescent cells and can be used as novel markers of senescence, such as arylsulfatase ARSA. The abundant secretome of senescent cells, known as SASP, is considered their main pathological mediator; however, little is known about the mechanisms of SASP secretion. Some secretory cells, including melanocytes, use the small GTPase RAB27A to perform lysosomal secretion. We found that this process is exacerbated in the case of senescent melanoma cells, as revealed by the exposure of lysosomal membrane integral proteins LAMP1 and LAMP2 in their plasma membrane. Interestingly, a subset of SASP components, including cytokines CCL2, CCL3, CXCL12, cathepsin CTSD, or the protease inhibitor SERPINE1, are secreted in a RAB27A-dependent manner in senescent melanoma cells. Finally, proteins previously identified as plasma biomarkers of aging are highly enriched in the lysosomes of senescent cells, including CTSD. We conclude that the lysosomal proteome of senescent cells is profoundly reconfigured, and that some senescent cells can be highly active in lysosomal exocytosis.
    Keywords:  SASP; aging; autophagy; cellular senescence; exocytosis; lysosome
  23. EMBO J. 2022 Sep 05. e110871
      Deubiquitylases (DUBs) are therapeutically amenable components of the ubiquitin machinery that stabilize substrate proteins. Their inhibition can destabilize oncoproteins that may otherwise be undruggable. Here, we screened for DUB vulnerabilities in multiple myeloma, an incurable malignancy with dependency on the ubiquitin proteasome system and identified OTUD6B as an oncogene that drives the G1/S-transition. LIN28B, a suppressor of microRNA biogenesis, is specified as a bona fide cell cycle-specific substrate of OTUD6B. Stabilization of LIN28B drives MYC expression at G1/S, which in turn allows for rapid S-phase entry. Silencing OTUD6B or LIN28B inhibits multiple myeloma outgrowth in vivo and high OTUD6B expression evolves in patients that progress to symptomatic multiple myeloma and results in an adverse outcome of the disease. Thus, we link proteolytic ubiquitylation with post-transcriptional regulation and nominate OTUD6B as a potential mediator of the MGUS-multiple myeloma transition, a central regulator of MYC, and an actionable vulnerability in multiple myeloma and other tumors with an activated OTUD6B-LIN28B axis.
    Keywords:  RNA binding proteins; cell cycle; deubiquitylases; multiple myloma; ubiquitin
  24. Proc Natl Acad Sci U S A. 2022 Sep 13. 119(37): e2201779119
      Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli. Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.
    Keywords:  Hsp70; HtpG; colibactin; heat stress; proteostasis
  25. Science. 2022 Sep 08. eabn5648
      Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. Here, we used genome-scale CRISPR screens to identify Lysosomal Enzyme Trafficking factor (LYSET) as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery, and mutations in LYSET can explain the phenotype of the associated disorder.
  26. Methods Cell Biol. 2022 ;pii: S0091-679X(22)00003-6. [Epub ahead of print]172 83-98
      Immunogenic cell death (ICD) is a modality of cellular demise that when it is induced by certain anticancer treatments can ignite an adaptive anticancer immune response. ICD is characterized by the emission of a specific set of danger-associated molecular patterns (DAMPs) including calreticulin exposure at the plasma membrane, ATP liberation, HMGB1 exodus and type-I IFN release. The apical signaling triggering the appearance of these hallmarks involves the phosphorylation on serine 51 of the α-subunit of eukaryotic initiation factor 2 (EIF2), a key protein in the orchestration of endoplasmic reticulum (ER) stress responses. EIF2α can be phosphorylated by a family of four EIF2A kinases: EIF2AK1-4 (best known as heme regulated inhibitor, HRI, protein kinase R, PKR, protein kinase R-like endoplasmic reticulum kinase, PERK, and general control non-derepressible 2, GCN2), that each respond to a specific type of cellular stress. Here, we describe different techniques to investigate the biochemical pathways leading to eIF2α phosphorylation in the context of ICD.
    Keywords:  Endoplasmic reticulum stress; Gene editing; Image analysis; Immunogenic cell death; eIF2α
  27. Methods Cell Biol. 2022 ;pii: S0091-679X(21)00132-1. [Epub ahead of print]172 99-114
      The exposure of calreticulin (CALR) on the cell surface of apoptotic cancer cells is an important "eat-me" signal that stimulates the engulfment by antigen presenting cells (APCs). When cells are exposed to immunogenic cell death (ICD) inducers, CALR translocates from the lumen of the endoplasmic reticulum (ER) to the cell surface, where it serves as a ligand for LDL-receptor-related protein 1 (LRP1, also known as CD91) expressed by dendritic cells (DCs). Surface-exposed CALR facilitates tumor antigen transfer to DCs and in turn antigen cross-presentation to cytotoxic T cells, altogether culminating in the activation of adaptive immune responses. Consistent with its role as an apical signaling event in anticancer immunity, blocking or neutralizing CALR abolishes the immune-dependent anticancer efficacy of a variety of ICD inducing anticancer agents. Recently we showed that saturating CALR receptors on DCs with abundant recombinant CALR protein, or soluble CALR secreted from cancer cells decreases the potency of ICD-mediated antitumor immune responses. Here we detail how to harness an artificially inducible release of soluble CALR from engineered cancer cells, which can blind DCs from recognizing immunogenic cancer cells, resulting in reduced anticancer immunity. This system offers precise control over the release of immunosuppressive soluble CALR, thus yielding a useful tool for the validation of ICD-inducing immunotherapies.
    Keywords:  DAMPs; Immunosuppression; Immunotherapy; RUSH
  28. iScience. 2022 Sep 16. 25(9): 104920
      The human brain consumes five orders of magnitude more energy than the sun by unit of mass and time. This staggering bioenergetic cost serves mostly synaptic transmission and actin cytoskeleton dynamics. The peak of both brain bioenergetic demands and the age of onset for neurodevelopmental disorders is approximately 5 years of age. This correlation suggests that defects in the machinery that provides cellular energy would be causative and/or consequence of neurodevelopmental disorders. We explore this hypothesis from the perspective of the machinery required for the synthesis of the electron transport chain, an ATP-producing and NADH-consuming enzymatic cascade. The electron transport chain is constituted by nuclear- and mitochondrial-genome-encoded subunits. These subunits are synthesized by the 80S and the 55S ribosomes, which are segregated to the cytoplasm and the mitochondrial matrix, correspondingly. Mitochondrial protein synthesis by the 55S ribosome is the rate-limiting step in the synthesis of electron transport chain components, suggesting that mitochondrial protein synthesis is a bottleneck for tissues with high bionergetic demands. We discuss genetic defects in the human nuclear and mitochondrial genomes that affect these protein synthesis machineries and cause a phenotypic spectrum spanning autism spectrum disorders to neurodegeneration during neurodevelopment. We propose that dysregulated mitochondrial protein synthesis is a chief, yet understudied, causative mechanism of neurodevelopmental and behavioral disorders.
    Keywords:  Biological Sciences; Cell Biology; Neuroscience
  29. Front Mol Biosci. 2022 ;9 982593
      The molecular chaperone Heat Shock Protein-90 (Hsp90) is known to interact with over 300 client proteins as well as regulatory factors (eg. nucleotide and proteins) that facilitate execution of its role as a chaperone and, ultimately, client protein activation. Hsp90 associates transiently with these molecular modulators during an eventful chaperone cycle, resulting in acquisition of flexible structural conformations, perfectly customized to the needs of each one of its client proteins. Due to the plethora and diverse nature of proteins it supports, the Hsp90 chaperone machinery is critical for normal cellular function particularly in response to stress. In diseases such as cancer, the Hsp90 chaperone machinery is hijacked for processes which encompass many of the hallmarks of cancer, including cell growth, survival, immune response evasion, migration, invasion, and angiogenesis. Elevated levels of extracellular Hsp90 (eHsp90) enhance tumorigenesis and the potential for metastasis. eHsp90 has been considered one of the new targets in the development of anti-cancer drugs as there are various stages of cancer progression where eHsp90 function could be targeted. Our limited understanding of the regulation of the eHsp90 chaperone machinery is a major drawback for designing successful Hsp90-targeted therapies, and more research is still warranted.
    Keywords:  ATP; Hsp90; MMP2; TIMP2; cancer therapy; co-chaperones; extracellular
  30. STAR Protoc. 2022 Sep 16. 3(3): 101654
      Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry. For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022).
    Keywords:  Cell biology; Gene expression; Molecular biology; Molecular/Chemical probes
  31. EMBO Mol Med. 2022 Sep 07. e16084
      Fn14 is a cell surface receptor with key functions in tissue homeostasis and injury but is also linked to chronic diseases. Despite its physiological and medical importance, the regulation of Fn14 signaling and turnover is only partly understood. Here, we demonstrate that Fn14 is cleaved within its transmembrane domain by the protease γ-secretase, resulting in secretion of the soluble Fn14 ectodomain (sFn14). Inhibition of γ-secretase in tumor cells reduced sFn14 secretion, increased full-length Fn14 at the cell surface, and enhanced TWEAK ligand-stimulated Fn14 signaling through the NFκB pathway, which led to enhanced release of the cytokine tumor necrosis factor. γ-Secretase-dependent sFn14 release was also detected ex vivo in primary tumor cells from glioblastoma patients, in mouse and human plasma and was strongly reduced in blood from human cancer patients dosed with a γ-secretase inhibitor prior to chimeric antigen receptor (CAR)-T-cell treatment. Taken together, our study demonstrates a novel function for γ-secretase in attenuating TWEAK/Fn14 signaling and suggests the use of sFn14 as an easily measurable pharmacodynamic biomarker to monitor γ-secretase activity in vivo.
    Keywords:  Alzheimer's disease; TNR12; ectodomain shedding; glioblastoma; intramembrane proteolysis
  32. Sci Rep. 2022 Sep 08. 12(1): 15201
      Multidomain proteins composed of individual domains connected by flexible linkers pose a challenge for structural studies due to their intrinsic conformational dynamics. Integrated modelling approaches provide a means to characterise protein flexibility by combining experimental measurements with molecular simulations. In this study, we characterise the conformational dynamics of the catalytic RBR domain of the E3 ubiquitin ligase HOIP, which regulates immune and inflammatory signalling pathways. Specifically, we combine small angle X-ray scattering experiments and molecular dynamics simulations to generate weighted conformational ensembles of the HOIP RBR domain using two different approaches based on maximum parsimony and maximum entropy principles. Both methods provide optimised ensembles that are instrumental in rationalising observed differences between SAXS-based solution studies and available crystal structures and highlight the importance of interdomain linker flexibility.