bims-proteo 2024-11-03 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–11–03
thirty-two papers selected by
Eric Chevet, INSERM

The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.
The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi.
Initiation of ERAD by the bifunctional complex of Mnl1 mannosidase and protein disulfide isomerase.
Ca+2 and Nε-lysine acetylation regulate the CALR-ATG9A interaction in the lumen of the endoplasmic reticulum.
Regulation of hepatic inclusions and fibrinogen biogenesis by SEL1L-HRD1 ERAD.
Senescence suppresses the integrated stress response and activates a stress-remodeled secretory phenotype.
IRGQ-mediated autophagy in MHC class I quality control promotes tumor immune evasion.
The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.
Caspase-2 is a condensate-mediated deubiquitinase in protein quality control.
Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design.
Molecular mechanisms of escrt-mediated autophagosome maturation in plants.
Limiting 20S proteasome assembly leads to unbalanced nucleo-cytoplasmic distribution of 26S/30S proteasomes and chronic proteotoxicity.
The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.
ER stress as a sentinel mechanism for ER Ca2+ homeostasis.
Structural and biophysical characterisation of ubiquitin variants that inhibit the ubiquitin conjugating enzyme Ube2d2.
Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors.
Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.
Selective regulation of aspartyl intramembrane protease activity by calnexin.
Liver CYP4A autophagic-lysosomal degradation (ALD): A major role for the autophagic receptor SQSTM1/p62 through an uncommon target interaction site.
The UPRising connection between endoplasmic reticulum stress and the tumor microenvironment.
The DNA damage response and RNA Polymerase II regulator Def1 has posttranscriptional functions in the cytoplasm.
AAA+ ATPase chaperone p97/VCPFAF2 governs basal pexophagy.
Using in vivo intact structure for system-wide quantitative analysis of changes in proteins.
BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection.
Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis.
The Hsp90 molecular chaperone as a global modifier of the genotype-phenotype-fitness map: An evolutionary perspective.
Dysregulation of FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis.
Exploring structural diversity across the protein universe with The Encyclopedia of Domains.
TMED4 facilitates Treg suppressive function via ROS homeostasis in tumor and autoimmune mouse models.
5'-tRNAGly(GCC) halves generated by IRE1α are linked to the ER stress response.
A genome-to-proteome atlas charts natural variants controlling proteome diversity and forecasts their fitness effects.
The protein segregase VCP/p97 promotes host antifungal defense via regulation of SYK activation.

bioRxiv. 2024 Oct 18. pii: 2024.10.17.618946. [Epub ahead of print]

The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.

Irina S Abaeva, Alexander G Bulakhov, Christopher U T Hellen, Tatyana V Pestova.

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome- associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2∼Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48- specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (Ub A ) and imposes K48-specificity by orienting Ub A such that its K48 is directly positioned to attack the thioester bond of the Ube2D1∼Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo .

DOI: https://doi.org/10.1101/2024.10.17.618946
Nat Commun. 2024 Oct 26. 15(1): 9257

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi.

Yannick Weyer, Sinead I Schwabl, Xuechen Tang, Astha Purwar, Konstantin Siegmann, Angela Ruepp, Theresia Dunzendorfer-Matt, Michael A Widerin, Veronika Niedrist, Noa J M Mutsters, Maria G Tettamanti, Sabine Weys, Bettina Sarg, Leopold Kremser, Klaus R Liedl, Oliver Schmidt, David Teis.

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

DOI: https://doi.org/10.1038/s41467-024-53676-6
bioRxiv. 2024 Oct 17. pii: 2024.10.17.618908. [Epub ahead of print]

Initiation of ERAD by the bifunctional complex of Mnl1 mannosidase and protein disulfide isomerase.

Dan Zhao, Xudong Wu, Tom A Rapoport.

Misfolded glycoproteins in the endoplasmic reticulum (ER) lumen are translocated into the cytosol and degraded by the proteasome, a conserved process called ER-associated protein degradation (ERAD). In S. cerevisiae , the glycan of these proteins is trimmed by the luminal mannosidase Mnl1 (Htm1) to generate a signal that triggers degradation. Curiously, Mnl1 is permanently associated with protein disulfide isomerase (Pdi1). Here, we have used cryo- electron microscopy, biochemical, and in vivo experiments to clarify how this complex initiates ERAD. The Mnl1-Pdi1 complex first de-mannosylates misfolded, globular proteins that are recognized through a C-terminal domain (CTD) of Mnl1; Pdi1 causes the CTD to ignore completely unfolded polypeptides. The disulfides of these globular proteins are then reduced by the Pdi1 component of the complex, generating unfolded polypeptides that can be translocated across the membrane. Mnl1 blocks the canonical oxidative function of Pdi1, but allows it to function as the elusive disulfide reductase in ERAD.

DOI: https://doi.org/10.1101/2024.10.17.618908
Sci Rep. 2024 10 26. 14(1): 25532

Ca+2 and Nε-lysine acetylation regulate the CALR-ATG9A interaction in the lumen of the endoplasmic reticulum.

Megan M Braun, Brendan K Sheehan, Samantha L Shapiro, Yun Ding, C Dustin Rubinstein, Brent P Lehman, Luigi Puglielli.

  The acetylation of autophagy protein 9 A (ATG9A) in the lumen of the endoplasmic reticulum (ER) by ATase1 and ATase2 regulates the induction of reticulophagy. Analysis of the ER-specific ATG9A interactome identified calreticulin (CALR), an ER luminal Ca+2-binding chaperone, as key for ATG9A activity. Specifically, if acetylated, ATG9A is sequestered by CALR and prevented from engaging FAM134B and SEC62. Under this condition, ATG9A is unable to activate the autophagy core machinery. In contrast, when non-acetylated, ATG9A is released by CALR and able to engage FAM134B and SEC62. In this study, we report that Ca+2 dynamics across the ER membrane regulate the ATG9A-CALR interaction as well as the ability of ATG9A to trigger reticulophagy. We show that the Ca+2-binding sites situated on the C-domain of CALR are essential for the ATG9A-CALR interaction. Finally, we show that K359 and K363 on ATG9A can influence the ATG9A-CALR interaction. Collectively, our results disclose a previously unidentified aspect of the complex mechanisms that regulate ATG9A activity. They also offer a possible area of intersection between Ca+2 metabolism, acetyl-CoA metabolism, and ER proteostasis.

Keywords:  ATG9A; Calcium; Calreticulin; Lysine acetylation; Proteostasis; Reticulophagy

DOI:  https://doi.org/10.1038/s41598-024-76854-4
Nat Commun. 2024 Oct 26. 15(1): 9244

Regulation of hepatic inclusions and fibrinogen biogenesis by SEL1L-HRD1 ERAD.

Zhenfeng Song, Pattaraporn Thepsuwan, Woosuk Steve Hur, Mauricio Torres, Shuangcheng Alivia Wu, Xiaoqiong Wei, Nusrat Jahan Tushi, Juncheng Wei, Francesca Ferraresso, Adrienne W Paton, James C Paton, Ze Zheng, Kezhong Zhang, Deyu Fang, Christian J Kastrup, Sunil Jaiman, Matthew James Flick, Shengyi Sun.

Impaired secretion of an essential blood coagulation factor fibrinogen leads to hepatic fibrinogen storage disease (HFSD), characterized by the presence of fibrinogen-positive inclusion bodies and hypofibrinogenemia. However, the molecular mechanisms underlying the biogenesis of fibrinogen in the endoplasmic reticulum (ER) remain unexplored. Here we uncover a key role of SEL1L-HRD1 complex of ER-associated degradation (ERAD) in the formation of aberrant inclusion bodies, and the biogenesis of nascent fibrinogen protein complex in hepatocytes. Acute or chronic deficiency of SEL1L-HRD1 ERAD in the hepatocytes leads to the formation of hepatocellular inclusion bodies. Proteomics studies followed by biochemical assays reveal fibrinogen as a major component of the inclusion bodies. Mechanistically, we show that the degradation of misfolded endogenous fibrinogen Aα, Bβ, and γ chains by SEL1L-HRD1 ERAD is indispensable for the formation of a functional fibrinogen complex in the ER. Providing clinical relevance of these findings, SEL1L-HRD1 ERAD indeed degrades and thereby attenuates the pathogenicity of two disease-causing fibrinogen γ mutants. Together, this study demonstrates an essential role of SEL1L-HRD1 ERAD in fibrinogen biogenesis and provides insight into the pathogenesis of protein-misfolding diseases.

DOI: https://doi.org/10.1038/s41467-024-53639-x
Mol Cell. 2024 Oct 24. pii: S1097-2765(24)00826-8. [Epub ahead of print]

Senescence suppresses the integrated stress response and activates a stress-remodeled secretory phenotype.

Matthew J Payea, Showkat A Dar, Carlos Anerillas, Jennifer L Martindale, Cedric Belair, Rachel Munk, Sulochan Malla, Jinshui Fan, Yulan Piao, Xiaoling Yang, Abid Rehman, Nirad Banskota, Kotb Abdelmohsen, Myriam Gorospe, Manolis Maragkakis.

  Senescence is a state of indefinite cell-cycle arrest associated with aging, cancer, and age-related diseases. Here, we find that translational deregulation, together with a corresponding maladaptive integrated stress response (ISR), is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response activating transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames (uORFs). Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. We also find that stress augments the senescence-associated secretory phenotype with sustained remodeling of inflammatory factors expression that is suppressed by non-uORF carrying ATF4 mRNA expression. Our results thus show that senescent cells possess a unique response to stress, which entails an increase in their inflammatory profile.

Keywords:  ATF4; ER stress; ISR; SASP; integrated stress response; nanopore direct RNA sequencing; proteomics; ribosome sequencing; senescence; senescence-associated secretory phenotype; translation

DOI:  https://doi.org/10.1016/j.molcel.2024.10.003
Cell. 2024 Oct 21. pii: S0092-8674(24)01148-6. [Epub ahead of print]

IRGQ-mediated autophagy in MHC class I quality control promotes tumor immune evasion.

Lina Herhaus, Uxía Gestal-Mato, Vinay V Eapen, Igor Mačinković, Henry J Bailey, Cristian Prieto-Garcia, Mohit Misra, Anne-Claire Jacomin, Aparna Viswanathan Ammanath, Ivan Bagarić, Jolina Michaelis, Joshua Vollrath, Ramachandra M Bhaskara, Georg Bündgen, Adriana Covarrubias-Pinto, Koraljka Husnjak, Jonathan Zöller, Ajami Gikandi, Sara Ribičić, Tobias Bopp, Gerbrand J van der Heden van Noort, Julian D Langer, Andreas Weigert, J Wade Harper, Joseph D Mancias, Ivan Dikic.

  The autophagy-lysosome system directs the degradation of a wide variety of cargo and is also involved in tumor progression. Here, we show that the immunity-related GTPase family Q protein (IRGQ), an uncharacterized protein to date, acts in the quality control of major histocompatibility complex class I (MHC class I) molecules. IRGQ directs misfolded MHC class I toward lysosomal degradation through its binding mode to GABARAPL2 and LC3B. In the absence of IRGQ, free MHC class I heavy chains do not only accumulate in the cell but are also transported to the cell surface, thereby promoting an immune response. Mice and human patients suffering from hepatocellular carcinoma show improved survival rates with reduced IRGQ levels due to increased reactivity of CD8+ T cells toward IRGQ knockout tumor cells. Thus, we reveal IRGQ as a regulator of MHC class I quality control, mediating tumor immune evasion.

Keywords:  GABARAPL2; IRGQ; LC3B; MHC class I; autophagy; hepatocellular carcinoma; immune evasion; quality control

DOI:  https://doi.org/10.1016/j.cell.2024.09.048
bioRxiv. 2024 Oct 17. pii: 2024.10.16.617214. [Epub ahead of print]

The Integrated Stress Response Suppresses PINK1-dependent Mitophagy by Preserving Mitochondrial Import Efficiency.

Mingchong Yang, Zengshuo Mo, Kelly Walsh, Wen Liu, Xiaoyan Guo.

Mitophagy is crucial for maintaining mitochondrial health, but how its levels adjust to different stress conditions remains unclear. In this study, we investigated the role of the DELE1-HRI axis of integrated stress response (ISR) in regulating mitophagy, a key mitochondrial stress pathway. Our findings show that the ISR suppresses mitophagy under non-depolarizing mitochondrial stress by positively regulating mitochondrial protein import, independent of ATF4 activation. Mitochondrial protein import is regulated by the rate of protein synthesis under both depolarizing and non-depolarizing stress. Without ISR, increased protein synthesis overwhelms the mitochondrial import machinery, reducing its efficiency. Under depolarizing stress, mitochondrial import is heavily impaired even with active ISR, leading to significant PINK1 accumulation. In contrast, non-depolarizing stress allows more efficient protein import in the presence of ISR, resulting in lower mitophagy. Without ISR, mitochondrial protein import becomes severely compromised, causing PINK1 accumulation to reach the threshold necessary to trigger mitophagy. These findings reveal a novel link between ISR-regulated protein synthesis, mitochondrial import, and mitophagy, offering potential therapeutic targets for diseases associated with mitochondrial dysfunction.

DOI: https://doi.org/10.1101/2024.10.16.617214
Nat Cell Biol. 2024 Oct 31.

Caspase-2 is a condensate-mediated deubiquitinase in protein quality control.

Yingwei Ge, Lijie Zhou, Yesheng Fu, Lijuan He, Yi Chen, Dingchang Li, Yuping Xie, Jun Yang, Haitao Wu, Hongmiao Dai, Zhiqiang Peng, Yong Zhang, Shaoqiong Yi, Bo Wu, Xin Zhang, Yangjun Zhang, Wantao Ying, Chun-Ping Cui, Cui Hua Liu, Lingqiang Zhang.

Protein ubiquitination plays a critical role in protein quality control in response to cellular stress. The excessive accumulation of ubiquitinated conjugates can be detrimental to cells and is recognized as a hallmark of multiple neurodegenerative diseases. However, an in-depth understanding of how the excessive ubiquitin chains are removed to maintain ubiquitin homeostasis post stress remains largely unclear. Here we found that caspase-2 (CASP2) accumulates in a ubiquitin and proteasome-positive biomolecular condensate, which we named ubstressome, following stress and functions as a deubiquitinase to remove overloaded ubiquitin chains on proteins prone to misfolding. Mechanistically, CASP2 binds to the poly-ubiquitinated conjugates through its allosteric ubiquitin-interacting motif-like region and decreases overloaded ubiquitin chains in a protease-dependent manner to promote substrate degradation. CASP2 deficiency in mice results in excessive accumulation of poly-ubiquitinated TAR DNA-binding protein 43, leading to motor defects. Our findings uncover a stress-evoked deubiquitinating activity of CASP2 in the maintenance of cellular ubiquitin homeostasis, which differs from the well-known roles of caspase in apoptosis and inflammation. These data also reveal unrecognized protein quality control functions of condensates in the removal of stress-induced ubiquitin chains.

DOI: https://doi.org/10.1038/s41556-024-01522-8
JACS Au. 2024 Oct 28. 4(10): 3857-3868

Non-Markovian Dynamic Models Identify Non-Canonical KRAS-VHL Encounter Complex Conformations for Novel PROTAC Design.

Yunrui Qiu, Rafal P Wiewiora, Jesus A Izaguirre, Huafeng Xu, Woody Sherman, Weiping Tang, Xuhui Huang.

Targeted protein degradation (TPD) is emerging as a promising therapeutic approach for cancer and other diseases, with an increasing number of programs demonstrating its efficacy in human clinical trials. One notable method for TPD is Proteolysis Targeting Chimeras (PROTACs) that selectively degrade a protein of interest (POI) through E3-ligase induced ubiquitination followed by proteasomal degradation. PROTACs utilize a warhead-linker-ligand architecture to bring the POI (bound to the warhead) and the E3 ligase (bound to the ligand) into proximity. The resulting non-native protein-protein interactions (PPIs) formed between the POI and E3 ligase lead to the formation of a stable ternary complex, enhancing cooperativity for TPD. A significant challenge in PROTAC design is the screening of the linkers to induce favorable non-native PPIs between POI and E3 ligase. Here, we present a physics-based computational protocol to predict noncanonical and metastable PPI interfaces between an E3 ligase and a given POI, aiding in the design of linkers to stabilize the ternary complex and enhance degradation. Specifically, we build the non-Markovian dynamic model using the Integrative Generalized Master equation (IGME) method from ∼1.5 ms all-atom molecular dynamics simulations of linker-less encounter complex, to systematically explore the inherent PPIs between the oncogene homologue protein and the von Hippel-Lindau E3 ligase. Our protocol revealed six metastable states each containing a different PPI interface. We selected three of these metastable states containing promising PPIs for linker design. Our selection criterion included thermodynamic and kinetic stabilities of PPIs and the accessibility between the solvent-exposed sites on the warheads and E3 ligand. One selected PPIs closely matches a recent cocrystal PPI interface structure induced by an experimentally designed PROTAC with potent degradation efficacy. We anticipate that our protocol has significant potential for widespread application in predicting metastable POI-ligase interfaces that can enable rational design of PROTACs.

DOI: https://doi.org/10.1021/jacsau.4c00503
Autophagy. 2024 Oct 30.

Molecular mechanisms of escrt-mediated autophagosome maturation in plants.

Niccolò Mosesso, Erika Isono.

  Diverse environmental stress factors affect the functionality of proteins and membrane compartments within cells causing potentially irremediable damage to the cell. A major process to eliminate nonfunctional molecular aggregates or damaged organelles under stress conditions is macroautophagy/autophagy, thus making its regulation critical for cellular adaptation and survival. The formation of autophagosomes is coordinated by a wide range of cellular factors and culminates in the closure of the cup-shaped double membrane or phagophore. The endosomal sorting complex required for transport (ESCRT) machinery has been proposed to mediate the sealing of the autophagic membranes. However, the molecular basis for ESCRT recruitment to phagophores under stress conditions are not yet fully understood. We recently described the role of ALIX (ALG-2 interacting protein-X) and its interactor CALB1 (Ca2+-dependent Lipid Binding protein 1) in autophagosome maturation during salt stress in Arabidopsis. Our study shows that CALB1 is important for phagophore closure and thus to the subsequent delivery to the vacuole. CALB1 localizes on salt-induced phagophores together with ALIX. CALB1 stimulates the phase separation of ALIX, which can facilitate the further ESCRT recruitment to phagophore membranes.

Keywords:  ALIX; Arabidopsis; C2 domain; ESCRT; molecular condensates; salt-induced autophagy

DOI:  https://doi.org/10.1080/15548627.2024.2423327
iScience. 2024 Nov 15. 27(11): 111095

Limiting 20S proteasome assembly leads to unbalanced nucleo-cytoplasmic distribution of 26S/30S proteasomes and chronic proteotoxicity.

Gabriel Ruiz-Romero, María Dolores Berdún, Mark Hochstrasser, Silvia Salas-Pino, Rafael R Daga.

  In addition to the degradation of cell-cycle proteins, short-lived, damaged, or unfolded proteins are constantly cleared from cells by the proteasome. During proliferation, the proteasome localizes to the nucleus and cytoplasm; however, the functional relevance of this compartmentalization remains unclear. Here, we show that folding stress increases 26S/30S proteasome activity, which correlates with the upregulation of Ump1, a chaperone involved in 20S assembly. Conversely, ump1 inactivation results in a drop of 20S and 26S/30S proteasomes. Limited 26S/30S proteasomes in ump1-deficient cells accumulate in the nucleus where they degrade mitotic substrates, allowing cells to proceed through mitosis; however, these cells present cytoplasmic aggregates and constitutive activation of the heat shock response. Thus, our data suggest that an increase in proteasome assembly induced by folding stress functions as an additional layer to proteasome regulation and highlight the importance of balanced proteasome compartmentalization to sustain cell proliferation while maintaining proper cytoplasmic proteostasis.

Keywords:  Functional aspects of cell biology; Molecular biology; Organizational aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2024.111095
bioRxiv. 2024 Oct 24. pii: 2024.10.24.620116. [Epub ahead of print]

The deubiquitinase Rpn11 functions as an allosteric ubiquitin sensor to promote substrate engagement by the 26S proteasome.

Zaw Min Htet, Ken C Dong, Andreas Martin.

The 26S proteasome is the major compartmental protease in eukaryotic cells, responsible for the ATP-dependent turnover of obsolete, damaged, or misfolded proteins that are delivered for degradation through attached ubiquitin modifications. In addition to targeting substrates to the proteasome, ubiquitin was recently shown to promote degradation initiation by directly modulating the conformational switching of the proteasome, yet the underlying mechanisms are unknown. Here, we used biochemical, mutational, and single-molecule FRET-based approaches to show that the proteasomal deubiquitinase Rpn11 functions as an allosteric sensor and facilitates the early steps of degradation. After substrate recruitment to the proteasome, ubiquitin binding to Rpn11 interferes with conformation-specific interactions of the ubiquitin-receptor subunit Rpn10, thereby stabilizing the engagement-competent state of the proteasome and expediting substrate insertion into the ATPase motor for mechanical translocation, unfolding, and Rpn11-mediated deubiquitination. These findings explain how modifications with poly-ubiquitin chains or multiple mono-ubiquitins allosterically promote substrate degradation and allow up to four-fold faster turnover by the proteasome.

DOI: https://doi.org/10.1101/2024.10.24.620116
Cell Calcium. 2024 Oct 18. pii: S0143-4160(24)00119-2. [Epub ahead of print]124 102961

ER stress as a sentinel mechanism for ER Ca2+ homeostasis.

Tadashi Makio, Junsheng Chen, Thomas Simmen.

  Endoplasmic reticulum (ER) stress is triggered upon the interference with oxidative protein folding that aims to produce fully folded, disulfide-bonded and glycosylated proteins, which are then competent to exit the ER. Many of the enzymes catalyzing this process require the binding of Ca2+ ions, including the chaperones BiP/GRP78, calnexin and calreticulin. The induction of ER stress with a variety of drugs interferes with chaperone Ca2+ binding, increases cytosolic Ca2+through the opening of ER Ca2+ channels, and activates store-operated Ca2+ entry (SOCE). Posttranslational modifications (PTMs) of the ER Ca2+ handling proteins through ER stress-dependent phosphorylation or oxidation control these mechanisms, as demonstrated in the case of the sarco/endoplasmic reticulum ATPase (SERCA), inositol 1,4,5 trisphosphate receptors (IP3Rs) or stromal interaction molecule 1 (STIM1). Their aim is to restore ER Ca2+ homeostasis but also to increase Ca2+ transfer from the ER to mitochondria during ER stress. This latter function boosts ER bioenergetics, but also triggers apoptosis if ER Ca2+ signaling persists. ER Ca2+ toolkit oxidative modifications upon ER stress can occur within the ER lumen or in the adjacent cytosol. Enzymes involved in this redox control include ER oxidoreductin 1 (ERO1) or the thioredoxin-family protein disulfide isomerases (PDI) and ERp57. A tight, but adaptive connection between ER Ca2+ content, ER stress and mitochondrial readouts allows for the proper functioning of many tissues, including skeletal muscle, the liver, and the pancreas, where ER stress either maintains or compromises their function, depending on its extent and context. Upon mutation of key regulators of ER Ca2+ signaling, diseases such as muscular defects (e.g., from mutated selenoprotein N, SEPN1/SELENON), or diabetes (e.g., from mutated PERK) are the result.

Keywords:  Endoplasmic reticulum (ER); Mitochondria; Mutation; Rare Disease

DOI:  https://doi.org/10.1016/j.ceca.2024.102961
FEBS J. 2024 Oct 29.

Structural and biophysical characterisation of ubiquitin variants that inhibit the ubiquitin conjugating enzyme Ube2d2.

Jeffery M R B McAlpine, Gene Zhu, Nicholas Pudjihartono, Joan Teyra, Michael J Currie, Zachary D Tillett, Renwick C J Dobson, Sachdev S Sidhu, Catherine L Day, Adam J Middleton.

  The ubiquitin-conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases, and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function relies on interactions with partner proteins, and the disruption of these is an effective way to modulate activity. Here, we report the discovery of ubiquitin variants (UbVs) that inhibit the E2 enzyme, Ube2d2 (UbcH5b). The six UbVs identified inhibit ubiquitin chain building, and the structural and biophysical characterisation of two of these demonstrate they bind to Ube2d2 with low micromolar affinity and high specificity. Both characterised UbVs bind at a site that overlaps with E1 binding, while the more inhibitory UbV has an additional binding site that blocks a critical non-covalent ubiquitin-binding site on the E2 enzyme. The discovery of novel protein-based ubiquitin derivatives that inhibit protein-protein interactions is an important step towards discovering small molecules that inhibit the activity of E2 enzymes. Furthermore, the specificity of the UbVs within the Ube2d family suggests that it may be possible to develop tools to selectively inhibit highly related E2 enzymes.

Keywords:  E2 enzyme; inhibitor; protein–protein interaction; ubiquitin; ubiquitin conjugating

DOI:  https://doi.org/10.1111/febs.17311
Mol Cancer. 2024 Oct 30. 23(1): 242

Tumour-intrinsic PDL1 signals regulate the Chk2 DNA damage response in cancer cells and mediate resistance to Chk1 inhibitors.

Clare E Murray, Anand V R Kornepati, Carlos Ontiveros, Yiji Liao, Bárbara de la Peña Avalos, Cody M Rogers, Zexuan Liu, Yilun Deng, Haiyan Bai, Suresh Kari, Alvaro S Padron, Jacob T Boyd, Ryan Reyes, Curtis A Clark, Robert S Svatek, Rong Li, Yanfen Hu, Meiling Wang, José R Conejo-Garcia, Lauren A Byers, Kavya Ramkumar, Anil K Sood, Jung-Min Lee, Christin E Burd, Ratna K Vadlamudi, Harshita B Gupta, Weixing Zhao, Eloïse Dray, Patrick Sung, Tyler J Curiel.

   BACKGROUND: Aside from the canonical role of PDL1 as a tumour surface-expressed immune checkpoint molecule, tumour-intrinsic PDL1 signals regulate non-canonical immunopathological pathways mediating treatment resistance whose significance, mechanisms, and therapeutic targeting remain incompletely understood. Recent reports implicate tumour-intrinsic PDL1 signals in the DNA damage response (DDR), including promoting homologous recombination DNA damage repair and mRNA stability of DDR proteins, but many mechanistic details remain undefined.
METHODS: We genetically depleted PDL1 from transplantable mouse and human cancer cell lines to understand consequences of tumour-intrinsic PDL1 signals in the DNA damage response. We complemented this work with studies of primary human tumours and inducible mouse tumours. We developed novel approaches to show tumour-intrinsic PDL1 signals in specific subcellular locations. We pharmacologically depleted tumour PDL1 in vivo in mouse models with repurposed FDA-approved drugs for proof-of-concept clinical translation studies.
RESULTS: We show that tumour-intrinsic PDL1 promotes the checkpoint kinase-2 (Chk2)-mediated DNA damage response. Intracellular but not surface-expressed PDL1 controlled Chk2 protein content post-translationally and independently of PD1 by antagonising PIRH2 E3 ligase-mediated Chk2 polyubiquitination and protein degradation. Genetic tumour PDL1 depletion specifically reduced tumour Chk2 content but not ATM, ATR, or Chk1 DDR proteins, enhanced Chk1 inhibitor (Chk1i) synthetic lethality in vitro in diverse human and murine tumour models, and improved Chk1i efficacy in vivo. Pharmacologic tumour PDL1 depletion with cefepime or ceftazidime replicated genetic tumour PDL1 depletion by reducing tumour Chk2, inducing Chk1i synthetic lethality in a tumour PDL1-dependent manner, and reducing in vivo tumour growth when combined with Chk1i.
CONCLUSIONS: Our data challenge the prevailing surface PDL1 paradigm, elucidate important and previously unappreciated roles for tumour-intrinsic PDL1 in regulating the ATM/Chk2 DNA damage response axis and E3 ligase-mediated protein degradation, suggest tumour PDL1 as a biomarker for Chk1i efficacy, and support the rapid clinical potential of pharmacologic tumour PDL1 depletion to treat selected cancers.

Keywords:  Chk2; DDR inhibitors; DNA damage repair; Immune checkpoints; PDL1; Synthetic lethality

DOI:  https://doi.org/10.1186/s12943-024-02147-z
J Cell Sci. 2024 Oct 28. pii: jcs.263548. [Epub ahead of print]

Activation of the mitochondrial unfolded protein response regulates the dynamic formation of stress granules.

Marta Lopez-Nieto, Zhaozhi Sun, Emily Relton, Rahme Safakli, Brian D Freibaum, J Paul Taylor, Alessia Ruggieri, Ioannis Smyrnias, Nicolas Locker.

  To rapidly adapt to harmful changes to their environment, cells activate the integrated stress response (ISR). This results in an adaptive transcriptional and translational rewiring, and the formation of biomolecular condensates named stress granules (SGs), to resolve stress. In addition to this first line of defence, the mitochondrial unfolded protein response (UPRmt) activates a specific transcriptional programme to maintain mitochondrial homeostasis. We present evidence that SGs and UPRmt pathways are intertwined and communicate. UPRmt induction results in eIF2a phosphorylation and the initial and transient formation of SGs, which subsequently disassemble. The induction of GADD34 during late UPRmt protects cells from prolonged stress by impairing further assembly of SGs. Furthermore, mitochondrial functions and cellular survival are enhanced during UPRmt activation when SGs are absent, suggesting that UPRmt-induced SGs have an adverse effect on mitochondrial homeostasis. These findings point to a novel crosstalk between SGs and the UPRmt that may contribute to restoring mitochondrial functions under stressful conditions.

Keywords:  GADD34; Integrated stress response; Mitochondrial stress response; Stress granules; UPRmt

DOI:  https://doi.org/10.1242/jcs.263548
Cell Mol Life Sci. 2024 Oct 26. 81(1): 441

Selective regulation of aspartyl intramembrane protease activity by calnexin.

Whendy Contreras, Jody Groenendyk, Marc Gentzel, Pascal Y Schönberg, Frank Buchholz, Marek Michalak, Bernd Schröder, Torben Mentrup.

  Signal peptide peptidase-like 2c (SPPL2c) is a testis-specific aspartyl intramembrane protease that contributes to male gamete function both by catalytic and non-proteolytic mechanisms. Here, we provide an unbiased characterisation of the in vivo interactome of SPPL2c identifying the ER chaperone calnexin as novel binding partner of this enzyme. Recruitment of calnexin specifically required the N-glycosylation within the N-terminal protease-associated domain of SPPL2c. Importantly, mutation of the single glycosylation site of SPPL2c or loss of calnexin expression completely prevented SPPL2c-mediated intramembrane proteolysis of all tested substrates. By contrast and despite rather promiscuous binding of calnexin to other SPP/SPPL proteases, expression of the chaperone was exclusively required for SPPL2c-mediated proteolysis. Despite some impact on the stability of SPPL2c most presumably due to assistance in folding of the luminal domain of the protease, calnexin appeared to be recruited rather constitutively to the protease thereby boosting its catalytic activity. In summary, we describe a novel, highly specific mode of intramembrane protease regulation, highlighting the need to systematically approach control mechanisms governing the proteolytic activity of other members of the aspartyl intramembrane protease family.

Keywords:  Calnexin; ER quality control; Intramembrane proteolysis; Male reproduction; Protease regulation; SPPL2c; Signal peptide peptidase

DOI:  https://doi.org/10.1007/s00018-024-05478-8
bioRxiv. 2024 Oct 14. pii: 2024.10.14.618315. [Epub ahead of print]

Liver CYP4A autophagic-lysosomal degradation (ALD): A major role for the autophagic receptor SQSTM1/p62 through an uncommon target interaction site.

Liang He, Doyoung Kwon, Michael J Trnka, Yi Liu, Jade Yang, Kathy Li, Rheem A Totah, Eric F Johnson, A L Burlingame, Maria Almira Correia.

The hepatic P450 hemoproteins CYPs 4A are typical N-terminally anchored Type I endoplasmic reticulum (ER)-proteins, that are inducible by hypolipidemic drugs and other "peroxisome proliferators". They are engaged in the ω-/ω-1-oxidation of various fatty acids including arachidonic acid, prostaglandins and leukotrienes and in the biotransformation of some therapeutic drugs. Herein we report that of the mammalian liver CYPs 4A, human CYP4A11 and mouse Cyp4a12a are preferential targets of the ER-lysosome-associated degradation (ERLAD). Consequently, these proteins are stabilized both as 1%Triton X100-soluble and -insoluble species in mouse hepatocytes and HepG2-cells deficient in the autophagic initiation ATG5-gene. Although these proteins exhibit surface LC3-interacting regions (LIRs) that would target them directly to the autophagosome, they nevertheless interact intimately with the autophagic receptor SQSTM1/p62. Through structural deletion analyses and site-directed mutagenesis, we have identified the Cyp4A-interacting p62 subdomain to lie between residues 170 and 233, which include its Traf6-binding and LIM-binding subdomains. Mice carrying a liver-specific genetic deletion of p62 residues 69-251 (p62Mut) that includes the CYP4A-interacting subdomain also exhibit Cyp4a-protein stabilization both as Triton X100-soluble and -insoluble species. Consistently, p62Mut mouse liver microsomes exhibit enhanced ω- and ω-1-hydroxylation of arachidonic acid to its physiologically active metabolites 19- and 20-HETEs relative to the corresponding wild-type mouse liver microsomes. Collectively, our findings suggest that any disruption of CYP4A ERLAD results in functionally active P450 protein and consequent production of proinflammatory metabolites on one hand, and insoluble aggregates on the other, which may contribute to pathological aggregates i.e. Mallory-Denk bodies/inclusions, hallmarks of many liver diseases.

DOI: https://doi.org/10.1101/2024.10.14.618315
Trends Cancer. 2024 Oct 29. pii: S2405-8033(24)00213-9. [Epub ahead of print]

The UPRising connection between endoplasmic reticulum stress and the tumor microenvironment.

Hery Urra, Raúl Aravena, Lucas González-Johnson, Claudio Hetz.

  The tumor microenvironment (TME) represents a dynamic network of cancer cells, stromal cells, immune mediators, and extracellular matrix components, crucial for cancer progression. Stress conditions such as oncogene activation, nutrient deprivation, and hypoxia disrupt the endoplasmic reticulum (ER), activating the unfolded protein response (UPR), the main adaptive mechanism to restore ER function. The UPR regulates cancer progression by engaging cell-autonomous and cell-non-autonomous mechanisms, reprogramming the stroma and promoting immune evasion, angiogenesis, and invasion. This review explores the role of UPR beyond cancer cells, focusing on how ER stress signaling reshapes the TME, supporting tumor growth. The therapeutic potential of targeting the UPR is also discussed.

Keywords:  angiogenesis; cancer; endoplasmic reticulum stress; tumor microenvironment; unfolded protein response

DOI:  https://doi.org/10.1016/j.trecan.2024.09.011
bioRxiv. 2024 Sep 16. pii: 2024.09.16.613278. [Epub ahead of print]

The DNA damage response and RNA Polymerase II regulator Def1 has posttranscriptional functions in the cytoplasm.

O T Akinniyi, S Kulkarni, M M Hribal, C A Keller, B Giardine, Joseph C Reese.

Yeast Def1 mediates RNA polymerase II degradation and transcription elongation during stress. Def1 is predominantly cytoplasmic, and DNA damage signals cause its proteolytic processing, liberating its N-terminus to enter the nucleus. Cytoplasmic functions for this abundant protein have not been identified. Proximity-labeling (BioID) experiments indicate that Def1 binds to an array of proteins involved in posttranscriptional control and translation of mRNAs. Deleting DEF1 reduces both mRNA synthesis and decay rates, indicating transcript buffering in the mutant. Directly tethering Def1 to a reporter mRNA suppressed expression, suggesting that Def1 directly regulates mRNAs. Surprisingly, we found that Def1 interacts with polyribosomes, which requires its ubiquitin-binding domain located in its N-terminus. The binding of Def1 to ribosomes requires the ubiquitylation of eS7a (Rsp7A) in the small subunit by the Not4 protein in the Ccr4-Not complex. Not4 ubiquitylation of the ribosome regulates translation quality control and co-translational mRNA decay. The polyglutamine-rich unstructured C-terminus of Def1 is required for its interaction with decay and translation factors, suggesting that Def1 acts as a ubiquitin-dependent scaffold to link translation status to mRNA decay. Thus, we have identified a novel function for this transcription and DNA damage response factor in posttranscriptional regulation in the cytoplasm and establish Def1 as a master regulator of gene expression, functioning during transcription, mRNA decay, and translation.

DOI: https://doi.org/10.1101/2024.09.16.613278
Nat Commun. 2024 Oct 29. 15(1): 9347

AAA+ ATPase chaperone p97/VCPFAF2 governs basal pexophagy.

Fumika Koyano, Koji Yamano, Tomoyuki Hoshina, Hidetaka Kosako, Yukio Fujiki, Keiji Tanaka, Noriyuki Matsuda.

Peroxisomes are organelles that are central to lipid metabolism and chemical detoxification. Despite advances in our understanding of peroxisome biogenesis, the mechanisms maintaining peroxisomal membrane proteins remain to be fully elucidated. We show here that mammalian FAF2/UBXD8, a membrane-associated cofactor of p97/VCP, maintains peroxisomal homeostasis by modulating the turnover of peroxisomal membrane proteins such as PMP70. In FAF2-deficient cells, PMP70 accumulation recruits the autophagy adaptor OPTN (Optineurin) to peroxisomes and promotes their autophagic clearance (pexophagy). Pexophagy is also induced by p97/VCP inhibition. FAF2 functions together with p97/VCP to negatively regulate pexophagy rather than as a factor for peroxisome biogenesis. Our results strongly suggest that p97/VCPFAF2-mediated extraction of ubiquitylated peroxisomal membrane proteins (e.g., PMP70) prevents peroxisomes from inducing nonessential autophagy under steady state conditions. These findings provide insight into molecular mechanisms underlying the regulation of peroxisomal integrity by p97/VCP and its associated cofactors.

DOI: https://doi.org/10.1038/s41467-024-53558-x
Nat Commun. 2024 Oct 29. 15(1): 9310

Using in vivo intact structure for system-wide quantitative analysis of changes in proteins.

Ahrum Son, Hyunsoo Kim, Jolene K Diedrich, Casimir Bamberger, Daniel B McClatchy, Stuart A Lipton, John R Yates.

Mass spectrometry-based methods can provide a global expression profile and structural readout of proteins in complex systems. Preserving the in vivo conformation of proteins in their innate state is challenging during proteomic experiments. Here, we introduce a whole animal in vivo protein footprinting method using perfusion of reagents to add dimethyl labels to exposed lysine residues on intact proteins which provides information about protein conformation. When this approach is used to measure dynamic structural changes during Alzheimer's disease (AD) progression in a mouse model, we detect 433 proteins that undergo structural changes attributed to AD, independent of aging, across 7 tissues. We identify structural changes of co-expressed proteins and link the communities of these proteins to their biological functions. Our findings show that structural alterations of proteins precede changes in expression, thereby demonstrating the value of in vivo protein conformation measurement. Our method represents a strategy for untangling mechanisms of proteostasis dysfunction caused by protein misfolding. In vivo whole-animal footprinting should have broad applicability for discovering conformational changes in systemic diseases and for the design of therapeutic interventions.

DOI: https://doi.org/10.1038/s41467-024-53582-x
PLoS Pathog. 2024 Oct 29. 20(10): e1012660

BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection.

Guillermo Najarro, Kevin Brackett, Hunter Woosley, Leah C Dorman, Vincent Turon-Lagot, Sudip Khadka, Catya Faeldonea, Osvaldo Kevin Moreno, Adriana Ramirez Negron, Christina Love, Ryan Ward, Charles Langelier, Frank McCarthy, Carlos Gonzalez, Joshua E Elias, Brooke M Gardner, Carolina Arias.

The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP's function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP's functions during infection by the Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies.

DOI: https://doi.org/10.1371/journal.ppat.1012660
Science. 2024 Nov;386(6721): 538-545

Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis.

Ziqiang P Li, Hortense Moreau, Jules D Petit, Tatiana S Moraes, Marija Smokvarska, Jessica Pérez-Sancho, Melina Petrel, Fanny Decoeur, Lysiane Brocard, Clément Chambaud, Magali S Grison, Andrea Paterlini, Marie Glavier, Lucie Hoornaert, Amit S Joshi, Etienne Gontier, William A Prinz, Yvon Jaillais, Antoine Taly, Felix Campelo, Marie-Cécile Caillaud, Emmanuelle M Bayer.

Diverging from conventional cell division models, plant cells undergo incomplete division to generate plasmodesmata communication bridges between daughter cells. Although fundamental for plant multicellularity, the molecular events leading to bridge stabilization, as opposed to severing, remain unknown. Using electron tomography, we mapped the transition from cell plate fenestrae to plasmodesmata. We show that the endoplasmic reticulum (ER) connects daughter cells across fenestrae, and as the cell plate matures, fenestrae contract, causing the plasma membrane (PM) to mold around constricted ER tubes. The ER's presence prevents fenestrae fusion, forming plasmodesmata, whereas its absence results in closure. The ER-PM protein tethers MCTP3, MCTP4, and MCTP6 further stabilize nascent plasmodesmata during fenestrae contraction. Genetic deletion in Arabidopsis reduces plasmodesmata formation. Our findings reveal how plants undergo incomplete division to promote intercellular communication.

DOI: https://doi.org/10.1126/science.adn4630
J Mol Biol. 2024 Oct 29. pii: S0022-2836(24)00475-3. [Epub ahead of print] 168846

The Hsp90 molecular chaperone as a global modifier of the genotype-phenotype-fitness map: An evolutionary perspective.

José Aguilar-Rodríguez, Christopher M Jakobson, Daniel F Jarosz.

  Global modifier genes influence the mapping of genotypes onto phenotypes and fitness through their epistatic interactions with genetic variants on a massive scale. The first such factor to be identified, Hsp90, is a highly conserved molecular chaperone that plays a central role in protein homeostasis. Hsp90 is a "hub of hubs" that chaperones proteins engaged in many key cellular and developmental regulatory networks. These clients, which are enriched in kinases, transcription factors, and E3 ubiquitin ligases, drive diverse cellular functions and are themselves highly connected. By contrast to many other hub proteins, the abundance and activity of Hsp90 changes substantially in response to shifting environmental conditions. As a result, Hsp90 modifies the functional impact of many genetic variants simultaneously in a manner that depends on environmental stress. Studies in diverse organisms suggest that this coupling between Hsp90 function and challenging environments exerts a substantial impact on what parts of the genome are visible to natural selection, expanding adaptive opportunities when most needed. In this Perspective, we explore the multifaceted role of Hsp90 as global modifier of the genotype-phenotype-fitness map as well as its implications for evolution in nature and the clinic.

Keywords:  epistasis; evolvability; molecular chaperones; molecular networks; mutational robustness

DOI:  https://doi.org/10.1016/j.jmb.2024.168846
bioRxiv. 2024 Oct 26. pii: 2024.10.25.620287. [Epub ahead of print]

Dysregulation of FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis.

Amanda K Casey, Nathan M Stewart, Naqi Zaidi, Hillery F Gray, Hazel A Fields, Masahiro Sakurai, Carlos A Pinzon-Arteaga, Bret M Evers, Jun Wu, Kim Orth.

Bi-functional enzyme FicD regulates the endoplasmic reticulum chaperone BiP using AMPylation and deAMPylation during ER homeostasis and stress, respectively. Human FicD with an arginine-to-serine mutation disrupts FicD deAMPylation activity resulting in severe neonatal diabetes. We generated the FicD R371S mutation in mice to create a pre-clinical murine model for neonatal diabetes. We observed elevated BiP AMPylation levels across multiple tissues and signature markers for diabetes including glucose intolerance and reduced serum insulin levels. While the pancreas of FicD R371S mice appeared normal at birth, adult FicD R371S mice displayed disturbed pancreatic islet organization that progressed with age. FicD R371S mice provide a preclinical mouse model for the study of UPR associated diabetes and demonstrate the essentiality of FicD for tissue resilience.

DOI: https://doi.org/10.1101/2024.10.25.620287
Science. 2024 Nov;386(6721): eadq4946

Exploring structural diversity across the protein universe with The Encyclopedia of Domains.

Andy M Lau, Nicola Bordin, Shaun M Kandathil, Ian Sillitoe, Vaishali P Waman, Jude Wells, Christine A Orengo, David T Jones.

The AlphaFold Protein Structure Database (AFDB) contains more than 214 million predicted protein structures composed of domains, which are independently folding units found in multiple structural and functional contexts. Identifying domains can enable many functional and evolutionary analyses but has remained challenging because of the sheer scale of the data. Using deep learning methods, we have detected and classified every domain in the AFDB, producing The Encyclopedia of Domains. We detected nearly 365 million domains, over 100 million more than can be found by sequence methods, covering more than 1 million taxa. Reassuringly, 77% of the nonredundant domains are similar to known superfamilies, greatly expanding representation of their domain space. We uncovered more than 10,000 new structural interactions between superfamilies and thousands of new folds across the fold space continuum.

DOI: https://doi.org/10.1126/science.adq4946
J Clin Invest. 2024 Oct 31. pii: e179874. [Epub ahead of print]

TMED4 facilitates Treg suppressive function via ROS homeostasis in tumor and autoimmune mouse models.

Zhenyan Jiang, Huizi Wang, Xiaoxia Wang, Hongrui Duo, Yuexiao Tao, Jia Li, Xin Li, Jiamin Liu, Jun Ni, Emily Jiatong Wu, Hongrui Xiang, Chenyang Guan, Xinyu Wang, Kun Zhang, Peng Zhang, Zhaoyuan Hou, Yong Liu, Zhengting Wang, Bing Su, Bo Li, Youjin Hao, Bin Li, Xuefeng Wu.

  Endoplasmic reticulum stress (ERS) plays crucial roles in maintaining regulatory T cells (Treg) stability and function, yet the underlying mechanism remains largely unexplored. Here we demonstrate that ERS-related protein transmembrane p24 trafficking protein 4 (TMED4) Treg-specific knockout (Tmed4ΔTreg) mice contain more Treg cells with impaired Foxp3 stability, Treg signature and suppressive activity, which leads to T cell hyperactivation, exacerbated inflammatory phenotype and boosted anti-tumor immunity in mice. Mechanistically, loss of Tmed4 causes defects in ERS and nuclear factor erythroid 2-related factor 2 (NRF2)-related antioxidant response, which results in excessive reactive oxygen species (ROS) that reduces Foxp3 stability and suppressive function of Treg cells in an IRE1α-XBP1 axis-dependent manner. The abnormalities can be effectively rescued by ROS scavenger, NRF2 inducer or forcible expression of IRE1α. Moreover, TMED4 suppresses IRE1α proteosome degradation via the ER-associated degradation (ERAD) system including BIP. Our study reveals that TMED4 maintains Treg cell stability and suppressive function through IRE1α-dependent ROS and the NRF2-related antioxidant response.

Keywords:  Adaptive immunity; Autoimmune diseases; Immunology; T cells

DOI:  https://doi.org/10.1172/JCI179874
Nat Commun. 2024 Oct 28. 15(1): 9273

5'-tRNAGly(GCC) halves generated by IRE1α are linked to the ER stress response.

Hanyong Jin, Ji-Hyun Yeom, Eunkyoung Shin, Yoonjie Ha, Haifeng Liu, Daeyoung Kim, Minju Joo, Yong-Hak Kim, Hak Kyun Kim, Minkyung Ryu, Hong-Man Kim, Jeongkyu Kim, Keun P Kim, Yoonsoo Hahn, Jeehyeon Bae, Kangseok Lee.

Transfer RNA halves (tRHs) have various biological functions. However, the biogenesis of specific 5'-tRHs under certain conditions remains unknown. Here, we report that inositol-requiring enzyme 1α (IRE1α) cleaves the anticodon stem-loop region of tRNAGly(GCC) to produce 5'-tRHs (5'-tRH-GlyGCC) with highly selective target discrimination upon endoplasmic reticulum (ER) stress. Levels of 5'-tRH-GlyGCC positively affect cancer cell proliferation and modulate mRNA isoform biogenesis both in vitro and in vivo; these effects require co-expression of two nuclear ribonucleoproteins, HNRNPM and HNRNPH2, which we identify as binding proteins of 5'-tRH-GlyGCC. In addition, under ER stress in vivo, we observe simultaneous induction of IRE1α and 5'-tRH-GlyGCC expression in mouse organs and a distantly related organism, Cryptococcus neoformans. Thus, collectively, our findings indicate an evolutionarily conserved function for IRE1α-generated 5'-tRH-GlyGCC in cellular adaptation upon ER stress.

DOI: https://doi.org/10.1038/s41467-024-53624-4
bioRxiv. 2024 Oct 22. pii: 2024.10.18.619054. [Epub ahead of print]

A genome-to-proteome atlas charts natural variants controlling proteome diversity and forecasts their fitness effects.

Christopher M Jakobson, Johannes Hartl, Pauline Trébulle, Michael Mülleder, Daniel F Jarosz, Markus Ralser.

Despite abundant genomic and phenotypic data across individuals and environments, the functional impact of most mutations on phenotype remains unclear. Here, we bridge this gap by linking genome to proteome in 800 meiotic progeny from an intercross between two closely related Saccharomyces cerevisiae isolates adapted to distinct niches. Modest genetic distance between the parents generated remarkable proteomic diversity that was amplified in the progeny and captured by 6,476 genotype-protein associations, over 1,600 of which we resolved to single variants. Proteomic adaptation emerged through the combined action of numerous cis - and trans -regulatory mutations, a regulatory architecture that was conserved across the species. Notably, trans -regulatory variants often arose in proteins not traditionally associated with gene regulation, such as enzymes. Moreover, the proteomic consequences of mutations predicted fitness under various stresses. Our study demonstrates that the collective action of natural genetic variants drives dramatic proteome diversification, with molecular consequences that forecast phenotypic outcomes.
Highlights: - Proteome diversity arises from natural genetic variants, with divergent proteomes in closely related parents and progeny. - Cis- regulatory elements had strong individual impacts, but coherent trans effects combined to dominate protein expression. - Directional selection and frequent transgression suggest much of the proteome is under selective pressure. - Many trans -regulators are enzymes or transporters, with fewer than 4% of pQTLs linking known interactors. - Genome-to-proteome connections predicted the fitness impact of mutations under various stresses, including a strong but hidden causal variant in IRA2/ NF1.

DOI: https://doi.org/10.1101/2024.10.18.619054
PLoS Pathog. 2024 Oct 29. 20(10): e1012674

The protein segregase VCP/p97 promotes host antifungal defense via regulation of SYK activation.

Zhugui Shao, Li Wang, Limin Cao, Tian Chen, Xin-Ming Jia, Wanwei Sun, Chengjiang Gao, Hui Xiao.

C-type lectin receptors (CLRs) are essential to execute host defense against fungal infection. Nevertheless, a comprehensive understanding of the molecular underpinnings of CLR signaling remains a work in progress. Here, we searched for yet-to-be-identified tyrosine-phosphorylated proteins in Dectin-1 signaling and linked the stress-response protein valosin containing protein (VCP)/p97 to Dectin-1 signaling. Knockdown of VCP expression or chemical inhibition of VCP's segregase activity dampened Dectin-1-elicited SYK activation in BMDMs and BMDCs, leading to attenuated expression of proinflammatory cytokines/chemokines such as TNF-α, IL-6 and CXCL1. Biochemical analyses demonstrated that VCP and its cofactor UFD1 form a complex with SYK and its phosphatase SHP-1 following Dectin-1 ligation, and knockdown of VCP led to a more prominent SYK and SHP-1 association. Further, SHP-1 became polyubiquitinated upon Dectin-1 activation, and VCP or UFD1 overexpression accelerated SHP-1 degradation. Conceivably, VCP may promote Dectin-1 signaling by pulling the ubiquitinated SHP-1 out of the SYK complex for degradation. Finally, genetic ablation of VCP in the neutrophil and macrophage compartment rendered the mice highly susceptible to infection by Candida albicans, an observation also phenocopied by administering the VCP inhibitor. These results collectively demonstrate that VCP is a previously unappreciated signal transducer of the Dectin-1 pathway and a crucial component of antifungal defense, and suggest a new mechanism regulating SYK activation.

DOI: https://doi.org/10.1371/journal.ppat.1012674