bims-proteo 2024-08-04 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–08–04
43 papers selected by
Eric Chevet, INSERM

Sestrin2 drives ER-phagy in response to protein misfolding.
ZDHHC7-mediated S-palmitoylation of ATG16L1 facilitates LC3 lipidation and autophagosome formation.
Ribosomal Frameshifting Selectively Modulates the Assembly, Function, and Pharmacological Rescue of a Misfolded CFTR Variant.
The ATP-driven extractor ATAD1/Msp1 proof-reads protein translocation into mitochondria.
Template-assisted covalent modification underlies activity of covalent molecular glues.
The IRE1α pathway in glomerular diseases: The unfolded protein response and beyond.
A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.
The V-ATPase/ATG16L1 axis is controlled by the V1H subunit.
Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2.
Benchmarking Methods for PROTAC Ternary Complex Structure Prediction.
Loss of transcriptional regulator of phospholipid biosynthesis alters post-translational modification of Sec61 translocon beta subunit Sbh1 in Saccharomyces cerevisiae.
HaloTag as a substrate-based macroautophagy reporter.
Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.
Small molecule FICD inhibitors suppress endogenous and pathologic FICD-mediated protein AMPylation.
Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells.
Targeted dephosphorylation of TFEB promotes its nuclear translocation.
Capturing the catalytic intermediates of parkin ubiquitination.
A modular DNA origami nanocompartment for engineering a cell-free, protein unfolding and degradation pathway.
IRE1 is implicated in protein synthesis regulation under ER stress conditions in plants.
Reconstituted cell-free translation systems for exploring protein folding and aggregation.
Floxuridine supports UPS independent of germline signaling and proteostasis regulators via involvement of detoxification in C. elegans.
An atypical form of 60S ribosomal subunit in Diamond-Blackfan anemia linked to RPL17 variants.
pyRBDome: a comprehensive computational platform for enhancing RNA-binding proteome data.
Golgi pH homeostasis stabilizes the lysosomal membrane through N-glycosylation of membrane proteins.
Deep learning guided design of dynamic proteins.
Engineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding.
Divergent folding-mediated epistasis among unstable membrane protein variants.
A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation.
De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.
Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells.
Rescuing error control in crosslinking mass spectrometry.
Eg5 UFMylation promotes spindle organization during mitosis.
Protocol for constructing tumor-targeting ANM-PROTACs for degradation of transcription factors.
Sodium arsenite induces aggresome formation by promoting PICK1 BAR domain homodimer formation.
Diffusing protein binders to intrinsically disordered proteins.
Proteostasis as a fundamental principle of Tau immunotherapy.
Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.
Massively parallel free energy calculations for in silico affinity maturation of designed miniproteins.
Dual-site molecular glues for enhancing protein-protein interactions of the CDK12-DDB1 complex.
Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.
Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury: PERK as a potential target for intervention.
Impact of protein and small molecule interactions on kinase conformations.
The influence of HLA genetic variation on plasma protein expression.

Dev Cell. 2024 Jul 30. pii: S1534-5807(24)00441-6. [Epub ahead of print]

Sestrin2 drives ER-phagy in response to protein misfolding.

Chiara De Leonibus, Marianna Maddaluno, Rosa Ferriero, Roberta Besio, Laura Cinque, Pei Jin Lim, Alessandro Palma, Rossella De Cegli, Salvatore Gagliotta, Sandro Montefusco, Maria Iavazzo, Marianne Rohrbach, Cecilia Giunta, Elena Polishchuk, Diego Louis Medina, Diego Di Bernardo, Antonella Forlino, Pasquale Piccolo, Carmine Settembre.

  Protein biogenesis within the endoplasmic reticulum (ER) is crucial for organismal function. Errors during protein folding necessitate the removal of faulty products. ER-associated protein degradation and ER-phagy target misfolded proteins for proteasomal and lysosomal degradation. The mechanisms initiating ER-phagy in response to ER proteostasis defects are not well understood. By studying mouse primary cells and patient samples as a model of ER storage disorders (ERSDs), we show that accumulation of faulty products within the ER triggers a response involving SESTRIN2, a nutrient sensor controlling mTORC1 signaling. SESTRIN2 induction by XBP1 inhibits mTORC1's phosphorylation of TFEB/TFE3, allowing these transcription factors to enter the nucleus and upregulate the ER-phagy receptor FAM134B along with lysosomal genes. This response promotes ER-phagy of misfolded proteins via FAM134B-Calnexin complex. Pharmacological induction of FAM134B improves clearance of misfolded proteins in ERSDs. Our study identifies the interplay between nutrient signaling and ER quality control, suggesting therapeutic strategies for ERSDs.

Keywords:  ER storage disorders; ER-phagy; FAM134B; TFEB; alpha(1)-antitrypsin Z (alpha(1)-ATZ); autophagy; collagen; endoplasmic reticulum; mTORC1; quality control

DOI:  https://doi.org/10.1016/j.devcel.2024.07.004
Autophagy. 2024 Aug 01.

ZDHHC7-mediated S-palmitoylation of ATG16L1 facilitates LC3 lipidation and autophagosome formation.

Fujing Wei, Yu Wang, Jia Yao, Ligang Mei, Xue Huang, Hesheng Kong, Jing Chen, Xiaorong Chen, Lu Liu, Zhuolin Wang, Jiaxin Wang, Jiong Song, Eryan Kong, Aimin Yang.

  Macroautophagy/autophagy is a fundamental cellular catabolic process that delivers cytoplasmic components into double-membrane vesicles called autophagosomes, which then fuse with lysosomes and their contents are degraded. Autophagy recycles cytoplasmic components, including misfolded proteins, dysfunctional organelles and even microbial invaders, thereby playing an essential role in development, immunity and cell death. Autophagosome formation is the main step in autophagy, which is governed by a set of ATG (autophagy related) proteins. ATG16L1 interacts with ATG12-ATG5 conjugate to form an ATG12-ATG5-ATG16L1 complex. The complex acts as a ubiquitin-like E3 ligase that catalyzes the lipidation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), which is crucial for autophagosome formation. In the present study, we found that ATG16L1 was subject to S-palmitoylation on cysteine 153, which was catalyzed by ZDHHC7 (zinc finger DHHC-type palmitoyltransferase 7). We observed that re-expressing ATG16L1 but not the S-palmitoylation-deficient mutant ATG16L1C153S rescued a defect in the lipidation of LC3 and the formation of autophagosomes in ATG16L1-KO (knockout) HeLa cells. Furthermore, increasing ATG16L1 S-palmitoylation by ZDHHC7 expression promoted the production of LC3-II, whereas reducing ATG16L1 S-palmitoylation by ZDHHC7 deletion inhibited the LC3 lipidation process and autophagosome formation. Mechanistically, the addition of a hydrophobic 16-carbon palmitoyl group on Cys153 residue of ATG16L1 enhances the formation of ATG16L1-WIPI2B complex and ATG16L1-RAB33B complex on phagophore, thereby facilitating the LC3 lipidation process and autophagosome formation. In conclusion, S-palmitoylation of ATG16L1 is essential for the lipidation process of LC3 and the formation of autophagosomes. Our research uncovers a new regulatory mechanism of ATG16L1 function in autophagy.

Keywords:  ATG16L1; LC3-II; ZDHHC7; autophagosome; autophagy; s-palmitoylation

DOI:  https://doi.org/10.1080/15548627.2024.2386915
bioRxiv. 2024 Jul 23. pii: 2023.05.02.539166. [Epub ahead of print]

Ribosomal Frameshifting Selectively Modulates the Assembly, Function, and Pharmacological Rescue of a Misfolded CFTR Variant.

Patrick Carmody, Francis J Roushar, Austin Tedman, Wei Wang, Madeline Herwig, Minsoo Kim, Eli Fritz McDonald, Karen Noguera, Jennifer Wong-Roushar, Jon-Luc Poirier, Nathan Zelt, Ben Pockrass, Andrew G McKee, Charles P Kuntz, S Vamsee Raju, Lars Plate, Wesley D Penn, Jonathan P Schlebach.

The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of cystic fibrosis (CF). The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex (EMC), which was previously found to modulate ribosome collisions during preemptive quality control of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.

DOI: https://doi.org/10.1101/2023.05.02.539166
Trends Cell Biol. 2024 Jul 31. pii: S0962-8924(24)00145-4. [Epub ahead of print]

The ATP-driven extractor ATAD1/Msp1 proof-reads protein translocation into mitochondria.

Maria Bohnert, Christos Gatsogiannis, Johannes M Herrmann.

  The accumulation of translocation intermediates in the mitochondrial import machinery threatens cellular fitness and is associated with cancer and neurodegeneration. A recent study by Weidberg and colleagues identifies ATAD1 as an ATP-driven extraction machine on the mitochondrial surface that pulls precursors into the cytosol to prevent clogging of mitochondrial import pores.

Keywords:  AAA protein; cancer; integrated stress response (ISR); mitochondria; protein import; quality control

DOI:  https://doi.org/10.1016/j.tcb.2024.07.007
Nat Chem Biol. 2024 Jul 29.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Yen-Der Li, Michelle W Ma, Muhammad Murtaza Hassan, Moritz Hunkeler, Mingxing Teng, Kedar Puvar, Justine C Rutter, Ryan J Lumpkin, Brittany Sandoval, Cyrus Y Jin, Anna M Schmoker, Scott B Ficarro, Hakyung Cheong, Rebecca J Metivier, Michelle Y Wang, Shawn Xu, Woong Sub Byun, Brian J Groendyke, Inchul You, Logan H Sigua, Isidoro Tavares, Charles Zou, Jonathan M Tsai, Paul M C Park, Hojong Yoon, Felix C Majewski, Haniya T Sperling, Jarrod A Marto, Jun Qi, Radosław P Nowak, Katherine A Donovan, Mikołaj Słabicki, Nathanael S Gray, Eric S Fischer, Benjamin L Ebert.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

DOI: https://doi.org/10.1038/s41589-024-01668-4
Front Mol Med. 2022 ;2 971247

The IRE1α pathway in glomerular diseases: The unfolded protein response and beyond.

José R Navarro-Betancourt, Andrey V Cybulsky.

  Endoplasmic reticulum (ER) function is vital for protein homeostasis ("proteostasis"). Protein misfolding in the ER of podocytes (glomerular visceral epithelial cells) is an important contributor to the pathogenesis of human glomerular diseases. ER protein misfolding causes ER stress and activates a compensatory signaling network called the unfolded protein response (UPR). Disruption of the UPR, in particular deletion of the UPR transducer, inositol-requiring enzyme 1α (IRE1α) in mouse podocytes leads to podocyte injury and albuminuria in aging, and exacerbates injury in glomerulonephritis. The UPR may interact in a coordinated manner with autophagy to relieve protein misfolding and its consequences. Recent studies have identified novel downstream targets of IRE1α, which provide new mechanistic insights into proteostatic pathways. Novel pathways of IRE1α signaling involve reticulophagy, mitochondria, metabolism, vesicular trafficking, microRNAs, and others. Mechanism-based therapies for glomerulopathies are limited, and development of non-invasive ER stress biomarkers, as well as targeting ER stress with pharmacological compounds may represent a therapeutic opportunity for preventing or attenuating progression of chronic kidney disease.

Keywords:  autophagy; cell injury; endoplasmic reticulum; glomerulonephitis; podocyte

DOI:  https://doi.org/10.3389/fmmed.2022.971247
Elife. 2024 Jul 29. pii: RP96979. [Epub ahead of print]13

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Joanne Tung, Lei Huang, Ginto George, Heather P Harding, David Ron, Adriana Ordonez.

  Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR.

Keywords:  ATF6⍺; CHO-K1 cells; CRT; UPR; calreticulin; cell biology; endoplasmic reticulum; genome-wide CRISPR/Cas9 screen; unfolded protein response

DOI:  https://doi.org/10.7554/eLife.96979
Mol Cell. 2024 Jul 24. pii: S1097-2765(24)00579-3. [Epub ahead of print]

The V-ATPase/ATG16L1 axis is controlled by the V1H subunit.

Lewis Timimi, Antoni G Wrobel, George N Chiduza, Sarah L Maslen, Antonio Torres-Méndez, Beatriz Montaner, Colin Davis, Taylor Minckley, Katriona L Hole, Andrea Serio, Michael J Devine, J Mark Skehel, John L Rubinstein, Anne Schreiber, Rupert Beale.

  Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.

Keywords:  ATG16L1; ATP6V1H; CASM; STING; V-ATPase; VAIL; autophagy; influenza; non-canonical autophagy; vacuolar ATPase

DOI:  https://doi.org/10.1016/j.molcel.2024.07.003
J Biol Chem. 2024 Jul 30. pii: S0021-9258(24)02117-3. [Epub ahead of print] 107616

Bivalent target-binding bioPROTACs induce potent degradation of oncogenic SHP2.

Megan Hoffman, David Krum, K Dane Wittrup.

  Targeted protein degradation is an emergent and rapidly evolving therapeutic strategy. In particular, biologics-based targeted degradation modalities (bioPROTACs) are relatively under explored compared to small molecules. Here, we investigate how target affinity, cellular localization, and valency of bioPROTACs impact efficacy of targeted degradation of the oncogenic phosphatase src-homology 2 containing protein tyrosine phosphatase-2 (SHP2). We identify bivalent recruitment of SHP2 by bioPROTACs as a broadly applicable strategy to improve potency. Moreover, we demonstrate that SHP2-targeted bioPROTACs can effectively counteract gain-of-function SHP2 mutants present in cancer, which are otherwise challenging to selectively target with small molecule constructs. Overall, this study demonstrates the utility of bioPROTACs for challenging targets, and further explicates design principles for therapeutic bioPROTACs.

Keywords:  BioPROTAC; Cancer; E3 Ubiquitin Ligase; PROTAC; Proteasome; Protein Degradation; Protein Engineering; SHP2; SPOP; Targeted Protein Degradation

DOI:  https://doi.org/10.1016/j.jbc.2024.107616
J Chem Inf Model. 2024 Aug 01.

Benchmarking Methods for PROTAC Ternary Complex Structure Prediction.

Evianne Rovers, Matthieu Schapira.

Proteolysis targeting chimeras (PROTACs) are bifunctional compounds that recruit an E3 ligase to a target protein to induce ubiquitination and degradation of the target. Rational optimization of PROTAC requires a structural model of the ternary complex. In the absence of an experimental structure, computational tools have emerged that attempt to predict PROTAC ternary complexes. Here, we systematically benchmark three commonly used tools: PRosettaC, MOE, and ICM. We find that these PROTAC-focused methods produce an array of ternary complex structures, including some that are observed experimentally, but also many that significantly deviate from the crystal structure. Molecular dynamics simulations show that PROTAC complexes may exist in a multiplicity of configurational states and question the use of experimentally observed structures as a reference for accurate predictions. The pioneering computational tools benchmarked here highlight the promises and challenges in the field and may be more valuable when guided by clear structural and biophysical data. The benchmarking data set that we provide may also be valuable for evaluating other and future computational tools for ternary complex modeling.

DOI: https://doi.org/10.1021/acs.jcim.4c00426
MicroPubl Biol. 2024 ;2024

Loss of transcriptional regulator of phospholipid biosynthesis alters post-translational modification of Sec61 translocon beta subunit Sbh1 in Saccharomyces cerevisiae.

Jacob M Miller, Mary E Tragesser-Tiña, Samantha M Turk, Eric M Rubenstein.

We recently discovered that disrupting phospholipid biosynthesis by eliminating the Ino2/4 transcriptional regulator impairs endoplasmic reticulum (ER)-associated degradation (ERAD) in Saccharomyces cerevisiae , but the mechanism is unclear. Phosphatidylcholine deficiency has been reported to accelerate degradation of Sec61 translocon beta subunit Sbh1 and ERAD cofactor Cue1. Here, we found that, unlike targeted phosphatidylcholine depletion, INO4 deletion does not destabilize Sbh1 or Cue1. However, we observed altered electrophoretic mobility of Sbh1 in ino4 Δ yeast, consistent with phospholipid-responsive post-translational modification. A better understanding of the molecular consequences of disrupted lipid homeostasis could lead to enhanced treatments for conditions associated with perturbed lipid biosynthesis.

DOI: https://doi.org/10.17912/micropub.biology.001260
Proc Natl Acad Sci U S A. 2024 Aug 06. 121(32): e2322500121

HaloTag as a substrate-based macroautophagy reporter.

Qiang Xiao, Gabrielle Cruz, Rachel Botham, Susan G Fox, Anan Yu, Seth Allen, Richard I Morimoto, Jeffery W Kelly.

  Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

Keywords:  HaloTag; autophagy; lysosome; macroautophagy; reporter

DOI:  https://doi.org/10.1073/pnas.2322500121
Proc Natl Acad Sci U S A. 2024 Aug 06. 121(32): e2319091121

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Gianna M Fote, Vinay V Eapen, Ryan G Lim, Clinton Yu, Lisa Salazar, Nicolette R McClure, Jharrayne McKnight, Thai B Nguyen, Marie C Heath, Alice L Lau, Mark A Villamil, Ricardo Miramontes, Ian H Kratter, Steven Finkbeiner, Jack C Reidling, Joao A Paulo, Peter Kaiser, Lan Huang, David E Housman, Leslie M Thompson, Joan S Steffan.

  Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Keywords:  Huntingtin; RNA-binding proteins; autophagy; ubiquitin; ubiquitin-binding domain

DOI:  https://doi.org/10.1073/pnas.2319091121
bioRxiv. 2024 Jul 16. pii: 2024.07.13.603377. [Epub ahead of print]

Small molecule FICD inhibitors suppress endogenous and pathologic FICD-mediated protein AMPylation.

Bhaskar K Chatterjee, Maroof Alam, Arghya Chakravorty, Shannon M Lacy, Jason Rech, Charles L Brooks, Peter D Arvan, Matthias C Truttmann.

  The AMP transferase, FICD, is an emerging drug target finetuning stress signaling in the endoplasmic reticulum (ER). FICD is a bi-functional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules to inhibit pathogenic FICD variants are lacking. Using an in-vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also efficiently inhibit pathogenic FICD variants and improve proinsulin processing in β cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.

Keywords:  AMPylation; BiP; FICD; cytotoxicity; high-throughput screen; proinsulin; small-molecule

DOI:  https://doi.org/10.1101/2024.07.13.603377
bioRxiv. 2024 Jul 26. pii: 2024.07.25.605204. [Epub ahead of print]

Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells.

Patrick J Nugent, Heungwon Park, Cynthia L Wladyka, Katharine Y Chen, Christine Bynum, Grace Quarterman, Andrew C Hsieh, Arvind Rasi Subramaniam.

RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.

DOI: https://doi.org/10.1101/2024.07.25.605204
iScience. 2024 Aug 16. 27(8): 110432

Targeted dephosphorylation of TFEB promotes its nuclear translocation.

Jin-Feng Zhao, Natalia Shpiro, Gajanan Sathe, Abigail Brewer, Thomas J Macartney, Nicola T Wood, Florentina Negoita, Kei Sakamoto, Gopal P Sapkota.

  Reversible phosphorylation of the transcription factor EB (TFEB) coordinates cellular responses to metabolic and other stresses. During nutrient replete and stressor-free conditions, phosphorylated TFEB is primarily localized to the cytoplasm. Stressor-mediated reduction of TFEB phosphorylation promotes its nuclear translocation and context-dependent transcriptional activity. In this study, we explored targeted dephosphorylation of TFEB as an approach to activate TFEB in the absence of nutrient deprivation or other cellular stress. Through an induction of proximity between TFEB and several phosphatases using the AdPhosphatase system, we demonstrate targeted dephosphorylation of TFEB in cells. Furthermore, by developing a heterobifunctional molecule BDPIC (bromoTAG-dTAG proximity-inducing chimera), we demonstrate targeted dephosphorylation of TFEB-dTAG through induced proximity to bromoTAG-PPP2CA. Targeted dephosphorylation of TFEB-dTAG by bromoTAG-PPP2CA with BDPIC at the endogenous levels is sufficient to induce nuclear translocation and some transcriptional activity of TFEB.

Keywords:  Health sciences; Medical specialty; Medicine; Precision medicine

DOI:  https://doi.org/10.1016/j.isci.2024.110432
Proc Natl Acad Sci U S A. 2024 Aug 06. 121(32): e2403114121

Capturing the catalytic intermediates of parkin ubiquitination.

Elizabeth M Connelly, Anne C Rintala-Dempsey, Mehmet Gundogdu, E Aisha Freeman, Joanna Koszela, Jacob D Aguirre, Grace Zhu, Outi Kämäräinen, Roya Tadayon, Helen Walden, Gary S Shaw.

  Parkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2~Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR-based chemical shift perturbation experiments. We show that a previously unidentified α-helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C-terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross-linking, and single turnover assays to establish and validate a model of full-length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity.

Keywords:  NMR; Parkinson’s disease; catalysis; parkin; protein structure

DOI:  https://doi.org/10.1073/pnas.2403114121
Nat Nanotechnol. 2024 Jul 29.

A modular DNA origami nanocompartment for engineering a cell-free, protein unfolding and degradation pathway.

J Huang, A Jaekel, J van den Boom, D Podlesainski, M Elnaggar, A Heuer-Jungemann, M Kaiser, H Meyer, B Saccà.

Within the cell, chemical reactions are often confined and organized through a modular architecture. This facilitates the targeted localization of molecular species and their efficient translocation to subsequent sites. Here we present a cell-free nanoscale model that exploits compartmentalization strategies to carry out regulated protein unfolding and degradation. Our synthetic model comprises two connected DNA origami nanocompartments (each measuring 25 nm × 41 nm × 53 nm): one containing the protein unfolding machine, p97, and the other housing the protease chymotrypsin. We achieve the unidirectional immobilization of p97 within the first compartment, establishing a gateway mechanism that controls substrate recruitment, translocation and processing within the second compartment. Our data show that, whereas spatial confinement increases the rate of the individual reactions by up to tenfold, the physical connection of the compartmentalized enzymes into a chimera efficiently couples the two reactions and reduces off-target proteolysis by almost sixfold. Hence, our modular approach may serve as a blueprint for engineering artificial nanofactories with reshaped catalytic performance and functionalities beyond those observed in natural systems.

DOI: https://doi.org/10.1038/s41565-024-01738-7
Plant Physiol Biochem. 2024 Jul 23. pii: S0981-9428(24)00631-4. [Epub ahead of print]215 108963

IRE1 is implicated in protein synthesis regulation under ER stress conditions in plants.

Jae Yong Yoo, Ki Seong Ko, Bich Ngoc Vu, Young Eun Lee, Ha Na Choi, Yoo Na Lee, Wahyu Indra Duwi Fanata, Rikno Harmoko, Sang-Kyu Lee, Woo Sik Chung, Jong Chan Hong, Kyun Oh Lee.

The unfolded protein response (UPR) is a crucial cellular mechanism for maintaining protein folding homeostasis during endoplasmic reticulum (ER) stress. In this study, the role of IRE1, a key component of the UPR, was investigated in protein translation regulation under ER stress conditions in Arabidopsis. We discovered that the loss of IRE1A and IRE1B leads to diminished protein translation, indicating a significant role for IRE1 in this process. However, this regulation was not solely dependent on the interaction with bZIP60, a key transcription factor in the UPR. Interestingly, while chemical chaperones TUDCA and PBA effectively alleviated the translation inhibition observed in ire1a ire1b mutants, this effect was more pronounced than the mitigation observed from suppressing GCN2 expression or introducing a non-phosphorylatable eIF2α variant. Additionally, the kinase and ribonuclease activities of IRE1B were demonstrated to be crucial for plant adaptation and protein synthesis regulation under ER stress conditions. Overall, this study not only highlights the complex regulatory mechanisms of IRE1 in plant ER stress responses but also provides insights into its multifaceted roles in protein translation regulation.

DOI: https://doi.org/10.1016/j.plaphy.2024.108963
J Mol Biol. 2024 Jul 27. pii: S0022-2836(24)00335-8. [Epub ahead of print] 168726

Reconstituted cell-free translation systems for exploring protein folding and aggregation.

Hideki Taguchi, Tatsuya Niwa.

  Protein folding is crucial for achieving functional three-dimensional structures. However, the process is often hampered by aggregate formation, necessitating the presence of chaperones and quality control systems within the cell to maintain protein homeostasis. Despite a long history of folding studies involving the denaturation and subsequent refolding of translation-completed purified proteins, numerous facets of cotranslational folding, wherein nascent polypeptides are synthesized by ribosomes and folded during translation, remain unexplored. Cell-free protein synthesis (CFPS) systems are invaluable tools for studying cotranslational folding, offering a platform not only for elucidating mechanisms but also for large-scale analyses to identify aggregation-prone proteins. This review provides an overview of the extensive use of CFPS in folding studies to date. In particular, we discuss a comprehensive aggregation formation assay of thousands of Escherichia coli proteins conducted under chaperone-free conditions using a reconstituted translation system, along with its derived studies.

Keywords:  PURE system; chaperone; cotranslational protein folding; protein aggregation; translation

DOI:  https://doi.org/10.1016/j.jmb.2024.168726
PLoS Genet. 2024 Jul 31. 20(7): e1011371

Floxuridine supports UPS independent of germline signaling and proteostasis regulators via involvement of detoxification in C. elegans.

Abhishek Anil Dubey, Anwesha Sarkar, Karolina Milcz, Natalia A Szulc, Pankaj Thapa, Małgorzata Piechota, Remigiusz A Serwa, Wojciech Pokrzywa.

The ubiquitin-proteasome system (UPS) is critical for maintaining proteostasis, influencing stress resilience, lifespan, and thermal adaptability in organisms. In Caenorhabditis elegans, specific proteasome subunits and activators, such as RPN-6, PBS-6, and PSME-3, are associated with heat resistance, survival at cold (4°C), and enhanced longevity at moderate temperatures (15°C). Previously linked to improving proteostasis, we investigated the impact of sterility-inducing floxuridine (FUdR) on UPS functionality under proteasome dysfunction and its potential to improve cold survival. Our findings reveal that FUdR significantly enhances UPS activity and resilience during proteasome inhibition or subunit deficiency, supporting worms' normal lifespan and adaptation to cold. Importantly, FUdR effect on UPS activity occurs independently of major proteostasis regulators and does not rely on the germ cells proliferation or spermatogenesis. Instead, FUdR activates a distinct detoxification pathway that supports UPS function, with GST-24 appearing to be one of the factors contributing to the enhanced activity of the UPS upon knockdown of the SKN-1-mediated proteasome surveillance pathway. Our study highlights FUdR unique role in the UPS modulation and its crucial contribution to enhancing survival under low-temperature stress, providing new insights into its mechanisms of action and potential therapeutic applications.

DOI: https://doi.org/10.1371/journal.pgen.1011371
JCI Insight. 2024 Aug 01. pii: e172475. [Epub ahead of print]

An atypical form of 60S ribosomal subunit in Diamond-Blackfan anemia linked to RPL17 variants.

Florence Fellmann, Carol Saunders, Marie-Françoise O'Donohue, David W Reid, Kelsey A McFadden, Nathalie Montel-Lehry, Cong Yu, Mingyan Fang, Jianguo Zhang, Beryl Royer-Bertrand, Pietro Farinelli, Narjesse Karboul, Jason R Willer, Lorraine Fievet, Zahurul Alam Bhuiyan, Alissa Lw Kleinhenz, Julie Jadeau, Joy Fulbright, Carlo Rivolta, Raffaele Renella, Nicholas Katsanis, Jacques S Beckmann, Christopher V Nicchitta, Lydie Da Costa, Erica E Davis, Pierre-Emmanuel Gleizes.

  Diamond-Blackfan anemia syndrome (DBA) is a ribosomopathy associated with loss-of-function variants in more than 20 ribosomal protein (RP) genes. Here, we report the genetic, functional and biochemical dissection of two multigenerational pedigrees with variants in RPL17, a large ribosomal subunit protein-encoding gene. Affected individuals had clinical features and erythroid proliferation defects consistent with DBA. Furthermore, RPL17/uL22 depletion resulted in anemia and micrognathia in zebrafish larvae, and in vivo complementation studies indicated that RPL17 variants were pathogenic. Lymphoblastoid cell lines (LCLs) derived from patients displayed a ribosomal RNA maturation defect reflecting haploinsufficiency of RPL17. The proteins encoded by RPL17 variants were not incorporated into ribosomes, but 10-20% of 60S ribosomal subunits contained a short form of 5.8S rRNA (5.8SC), a species that is marginal in normal cells. These atypical 60S subunits were actively engaged in translation. Ribosome profiling showed changes of the translational profile, but those are similar to LCLs bearing RPS19 variants. These results link an additional RP gene to DBA. They show that ribosomes can be modified substantially by RPL17 haploinsufficiency, but support the paradigm that translation alterations in DBA are primarily related to insufficient ribosome production rather than to changes in ribosome structure or composition.

Keywords:  Bone marrow; Genetics; Hematology; RNA processing; Translation

DOI:  https://doi.org/10.1172/jci.insight.172475
Life Sci Alliance. 2024 Oct;pii: e202402787. [Epub ahead of print]7(10):

pyRBDome: a comprehensive computational platform for enhancing RNA-binding proteome data.

Liang-Cui Chu, Niki Christopoulou, Hugh McCaughan, Sophie Winterbourne, Davide Cazzola, Shichao Wang, Ulad Litvin, Salomé Brunon, Patrick Jb Harker, Iain McNae, Sander Granneman.

High-throughput proteomics approaches have revolutionised the identification of RNA-binding proteins (RBPome) and RNA-binding sequences (RBDome) across organisms. Yet, the extent of noise, including false positives, associated with these methodologies, is difficult to quantify as experimental approaches for validating the results are generally low throughput. To address this, we introduce pyRBDome, a pipeline for enhancing RNA-binding proteome data in silico. It aligns the experimental results with RNA-binding site (RBS) predictions from distinct machine-learning tools and integrates high-resolution structural data when available. Its statistical evaluation of RBDome data enables quick identification of likely genuine RNA-binders in experimental datasets. Furthermore, by leveraging the pyRBDome results, we have enhanced the sensitivity and specificity of RBS detection through training new ensemble machine-learning models. pyRBDome analysis of a human RBDome dataset, compared with known structural data, revealed that although UV-cross-linked amino acids were more likely to contain predicted RBSs, they infrequently bind RNA in high-resolution structures. This discrepancy underscores the limitations of structural data as benchmarks, positioning pyRBDome as a valuable alternative for increasing confidence in RBDome datasets.

DOI: https://doi.org/10.26508/lsa.202402787
Life Sci Alliance. 2024 Oct;pii: e202402677. [Epub ahead of print]7(10):

Golgi pH homeostasis stabilizes the lysosomal membrane through N-glycosylation of membrane proteins.

Yu-Shin Sou, Junji Yamaguchi, Keisuke Masuda, Yasuo Uchiyama, Yusuke Maeda, Masato Koike.

Protein glycosylation plays a vital role in various cellular functions, many of which occur within the Golgi apparatus. The Golgi pH regulator (GPHR) is essential for the proper functioning of the Golgi apparatus. The lysosomal membrane contains highly glycosylated membrane proteins in abundance. This study investigated the role of the Golgi luminal pH in N-glycosylation of lysosomal membrane proteins and the effect of this protein modification on membrane stability using Gphr-deficient MEFs. We showed that Gphr deficiency causes an imbalance in the Golgi luminal pH, resulting in abnormal protein N-glycosylation, indicated by a reduction in sialylated glycans and markedly reduced molecular weight of glycoproteins. Further experiments using FRAP and PLA revealed that Gphr deficiency prevented the trafficking dynamics and proximity condition of glycosyltransferases in the Golgi apparatus. In addition, incomplete N-glycosylation of lysosomal membrane proteins affected lysosomal membrane stability, as demonstrated by the increased susceptibility to lysosomal damage. Thus, this study highlights the critical role of Golgi pH regulation in controlling protein glycosylation and the impact of Golgi dysfunction on lysosomal membrane stability.

DOI: https://doi.org/10.26508/lsa.202402677
bioRxiv. 2024 Jul 19. pii: 2024.07.17.603962. [Epub ahead of print]

Deep learning guided design of dynamic proteins.

Amy B Guo, Deniz Akpinaroglu, Mark J S Kelly, Tanja Kortemme.

Deep learning has greatly advanced design of highly stable static protein structures, but the controlled conformational dynamics that are hallmarks of natural switch-like signaling proteins have remained inaccessible to de novo design. Here, we describe a general deep-learning-guided approach for de novo design of dynamic changes between intra-domain geometries of proteins, similar to switch mechanisms prevalent in nature, with atom-level precision. We solve 4 structures validating the designed conformations, show microsecond transitions between them, and demonstrate that the conformational landscape can be modulated by orthosteric ligands and allosteric mutations. Physics-based simulations are in remarkable agreement with deep-learning predictions and experimental data, reveal distinct state-dependent residue interaction networks, and predict mutations that tune the designed conformational landscape. Our approach demonstrates that new modes of motion can now be realized through de novo design and provides a framework for constructing biology-inspired, tunable and controllable protein signaling behavior de novo.

DOI: https://doi.org/10.1101/2024.07.17.603962
Nat Commun. 2024 Aug 02. 15(1): 6511

Engineering of a mammalian VMAT2 for cryo-EM analysis results in non-canonical protein folding.

Ying Lyu, Chunting Fu, Haiyun Ma, Zhaoming Su, Ziyi Sun, Xiaoming Zhou.

Vesicular monoamine transporter 2 (VMAT2) belongs to the major facilitator superfamily (MFS), and mediates cytoplasmic monoamine packaging into presynaptic vesicles. Here, we present two cryo-EM structures of VMAT2, with a frog VMAT2 adopting a canonical MFS fold and an engineered sheep VMAT2 adopting a non-canonical fold. Both VMAT2 proteins mediate uptake of a selective fluorescent VMAT2 substrate into cells. Molecular docking, substrate binding and transport analysis reveal potential substrate binding mechanism in VMAT2. Meanwhile, caution is advised when interpreting engineered membrane protein structures.

DOI: https://doi.org/10.1038/s41467-024-50934-5
Elife. 2024 Jul 30. pii: RP92406. [Epub ahead of print]12

Divergent folding-mediated epistasis among unstable membrane protein variants.

Laura M Chamness, Charles P Kuntz, Andrew G McKee, Wesley D Penn, Christopher M Hemmerich, Douglas B Rusch, Hope Woods, Dyotima, Jens Meiler, Jonathan P Schlebach.

  Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

Keywords:  epistasis; human; membrane protein folding; molecular biophysics; protein evolution; protein misfolding; structural biology; translocon

DOI:  https://doi.org/10.7554/eLife.92406
bioRxiv. 2024 Jul 23. pii: 2024.07.22.604634. [Epub ahead of print]

A spectrum of clinically-identified cysteine mutations in fibulin-3 (EFEMP1) highlight its disulfide bonding complexity and potential to induce stress response activation.

Gracen E Collier, John D Hulleman.

Fibulin-3 (FBLN3), also known as EFEMP1, is a secreted extracellular matrix (ECM) glycoprotein that contains forty cysteine residues. These cysteines, which are distributed across one atypical and five canonical calcium-binding epidermal growth factor (EGF) domains, are important for regulating FBLN3 structure, secretion, and presumably function. As evidence of this importance, a rare homozygous p.C55R mutation in FBLN3 negates its function, alters disulfide bonding, and causes marfanoid syndrome. Additional studies suggest that heterozygous premature stop codon mutations in FBLN3 may also cause similar, albeit less severe, connective tissue disorders. Interestingly, a series of twenty-four cysteine mutations in FBLN3 have been identified in the human population and published in the Clinical Variation (ClinVar) and gnomAD databases. We tested how seven of these cysteine mutants (five loss-of-cysteine variants: C42Y, C190R, C218R, C252F, and C365S, two gain-of-cysteine variants: R358C, Y369C) and two newly developed mutations (G57C and Y397C) altered FBLN3 secretion, disulfide bonding, MMP2 zymography, and stress response activation Surprisingly, we found a wide variety of biochemical behaviors: i) loss-of-cysteine variants correlated with an increased likelihood of disulfide dimer formation, ii) N-terminal mutations were less likely to disrupt secretion, and were less prone to aggregation, iii) in contrast to wild-type FBLN3, multiple, but not all variants failed to induce MMP2 levels in cell culture, and iv) C-terminal mutations (either loss or gain of cysteines) were more prone to significant secretion defects, intracellular accumulation/misfolding, and stress response activation. These results provide molecular and biochemical insight into FBLN3 folding, secretion, and function for many cysteine mutations found in the human population, some of which may increase the likelihood of subclinical connective tissue or other FBLN3-associated haploinsufficiency diseases.

DOI: https://doi.org/10.1101/2024.07.22.604634
bioRxiv. 2024 Jul 22. pii: 2023.06.26.546591. [Epub ahead of print]

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Suhaas Bhat, Kalyan Palepu, Lauren Hong, Joey Mao, Tianzheng Ye, Rema Iyer, Lin Zhao, Tianlai Chen, Sophia Vincoff, Rio Watson, Tian Wang, Divya Srijay, Venkata Srikar Kavirayuni, Kseniia Kholina, Shrey Goel, Pranay Vure, Aniruddha J Desphande, Scott H Soderling, Matthew P DeLisa, Pranam Chatterjee.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Pep tide Pr ioritization via CLIP ( PepPrCLIP ) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro . Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.

DOI: https://doi.org/10.1101/2023.06.26.546591
STAR Protoc. 2024 Jul 31. pii: S2666-1667(24)00291-0. [Epub ahead of print]5(3): 103126

Protocol for analyzing TRAIL- and Fas-induced signaling complexes by immunoprecipitation from human cells.

Pavel Davidovich, Seamus J Martin.

  Engagement of TRAIL or Fas death receptors can trigger the assembly of cytoplasmic caspase-8/FADD/RIPK1 (FADDosome) signaling complexes that promote nuclear factor κB (NF-κB) activation. Here, we present a protocol for immunoprecipitation of TRAIL- or Fas-induced FADDosomes from human cell lines. We describe steps for stimulating human cells with TRAIL or Fas ligand, followed by preparation of membrane death receptor-associated, as well as cytoplasmic FADDosome, signaling complexes. This protocol has application in the analysis of death receptor-induced signaling complex formation. For complete details on the use and execution of this protocol, please refer to Davidovich et al.1.

Keywords:  cell biology; immunology; protein biochemistry

DOI:  https://doi.org/10.1016/j.xpro.2024.103126
Mol Syst Biol. 2024 Aug 02.

Rescuing error control in crosslinking mass spectrometry.

Lutz Fischer, Juri Rappsilber.

  Crosslinking mass spectrometry is a powerful tool to study protein-protein interactions under native or near-native conditions in complex mixtures. Through novel search controls, we show how biassing results towards likely correct proteins can subtly undermine error estimation of crosslinks, with significant consequences. Without adjustments to address this issue, we have misidentified an average of 260 interspecies protein-protein interactions across 16 analyses in which we synthetically mixed data of different species, misleadingly suggesting profound biological connections that do not exist. We also demonstrate how data analysis procedures can be tested and refined to restore the integrity of the decoy-false positive relationship, a crucial element for reliably identifying protein-protein interactions.

Keywords:  Crosslinking Mass Spectrometry; Data Analysis; Data Reliability; Error Estimation; Proteomics

DOI:  https://doi.org/10.1038/s44320-024-00057-2
Cell Death Dis. 2024 Jul 31. 15(7): 544

Eg5 UFMylation promotes spindle organization during mitosis.

Guangxu Li, Yuanjiang Huang, Wenbo Han, Liyi Wei, Hongjing Huang, Yingbao Zhu, Qiao Xiao, Zujia Wang, Wen Huang, Ranhui Duan.

UFMylation is a highly conserved ubiquitin-like post-translational modification that catalyzes the covalent linkage of UFM1 to its target proteins. This modification plays a critical role in the maintenance of endoplasmic reticulum proteostasis, DNA damage response, autophagy, and transcriptional regulation. Mutations in UFM1, as well as in its specific E1 enzyme UBA5 and E2 enzyme UFC1, have been genetically linked to microcephaly. Our previous research unveiled the important role of UFMylation in regulating mitosis. However, the underlying mechanisms have remained unclear due to the limited identification of substrates. In this study, we identified Eg5, a motor protein crucial for mitotic spindle assembly and maintenance, as a novel substrate for UFMylation and identified Lys564 as the crucial UFMylation site. UFMylation did not alter its transcriptional level, phosphorylation level, or protein stability, but affected the mono-ubiquitination of Eg5. During mitosis, Eg5 and UFM1 co-localize at the centrosome and spindle apparatus, and defective UFMylation leads to diminished spindle localization of Eg5. Notably, the UFMylation-defective Eg5 mutant (K564R) exhibited shorter spindles, metaphase arrest, spindle checkpoint activation, and a failure of cell division in HeLa cells. Overall, Eg5 UFMylation is essential for proper spindle organization, mitotic progression, and cell proliferation.

DOI: https://doi.org/10.1038/s41419-024-06934-w
STAR Protoc. 2024 Jul 27. pii: S2666-1667(24)00385-X. [Epub ahead of print]5(3): 103220

Protocol for constructing tumor-targeting ANM-PROTACs for degradation of transcription factors.

Jin Li, Chao Liang.

  AS1411-NCL-MDM2-based proteolysis-targeting chimeras (ANM-PROTACs) are capable of inducing selective degradation of transcription factors (TFs) in tumor cells. Here, we present a protocol for constructing ANM-PROTACs. We describe steps for molecular design of the ANM-PROTACs, assembly and characterization of the ANM-PROTACs, and initial assessment of in vitro TF degradation potency. We then detail procedures for validation of selective degradation of TFs via proteomic analysis. This protocol has been successfully applied to degrade various TFs across multiple tumor cell lines. For complete details on the use and execution of this protocol, please refer to Fu et al.1.

Keywords:  Cancer; Mass Spectrometry; Protein Biochemistry; Proteomics

DOI:  https://doi.org/10.1016/j.xpro.2024.103220
Mol Biol Cell. 2024 Jul 31. mbcE24050201

Sodium arsenite induces aggresome formation by promoting PICK1 BAR domain homodimer formation.

John Ho Chun Lai, Marianthi Tsogka, Jun Xia.

The aggresome is a perinuclear structure that sequesters misfolded proteins. It's implicated in various neurodegenerative diseases. The perinuclear structure enriched with PICK1 was found to be inducible by cellular stressors, colocalizing with microtubule-organizing center (MTOC) markers and ubiquitin, hence classifying it as an aggresome. Sodium arsenite but not arsenate was found to potently induce aggresome formation through an integrated stress response (ISR)-independent pathway. In HEK293T cells, under arsenite stress, PICK1 localization to the aggresome was prioritized, and formation of PICK1 homodimers was favored. Additionally, PICK1 could enhance protein entry into aggresomes. This study shows that arsenite can induce the formation of both RNA stress granules and aggresomes at the same time, and that PICK1 shows conditional localization to aggresomes, suggesting a possible involvement of PICK1 in neurodegenerative diseases. [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E24-05-0201
bioRxiv. 2024 Jul 16. pii: 2024.07.16.603789. [Epub ahead of print]

Diffusing protein binders to intrinsically disordered proteins.

Caixuan Liu, Kejia Wu, Hojun Choi, Hannah Han, Xulie Zhang, Joseph L Watson, Sara Shijo, Asim K Bera, Alex Kang, Evans Brackenbrough, Brian Coventry, Derrick R Hick, Andrew N Hoofnagle, Ping Zhu, Xingting Li, Justin Decarreau, Stacey R Gerben, Wei Yang, Xinru Wang, Mila Lamp, Analisa Murray, Magnus Bauer, David Baker.

  Proteins which bind intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) with high affinity and specificity could have considerable utility for therapeutic and diagnostic applications. However, a general methodology for targeting IDPs/IDRs has yet to be developed. Here, we show that starting only from the target sequence of the input, and freely sampling both target and binding protein conformation, RFdiffusion can generate binders to IDPs and IDRs in a wide range of conformations. We use this approach to generate binders to the IDPs Amylin, C-peptide and VP48 in a range of conformations with Kds in the 3 -100nM range. The Amylin binder inhibits amyloid fibril formation and dissociates existing fibers, and enables enrichment of amylin for mass spectrometry-based detection. For the IDRs G3bp1, common gamma chain (IL2RG) and prion, we diffused binders to beta strand conformations of the targets, obtaining 10 to 100 nM affinity. The IL2RG binder colocalizes with the receptor in cells, enabling new approaches to modulating IL2 signaling. Our approach should be widely useful for creating binders to flexible IDPs/IDRs spanning a wide range of intrinsic conformational preferences.

Keywords:  Amyloid fibril dissociation; Intrinsically disordered protein; RFdiffusion; Rosetta; deep learning; diagnostics; intrinsically disordered region; protein design

DOI:  https://doi.org/10.1101/2024.07.16.603789
Brain. 2024 Jul 29. pii: awae254. [Epub ahead of print]

Proteostasis as a fundamental principle of Tau immunotherapy.

Esteban Cruz, Rebecca M Nisbet, Pranesh Padmanabhan, Ashley J van Waardenberg, Mark E Graham, Godfrey Nkajja, Swara Tapaswi, Bradley J Connor, Phil Robinson, Jürgen Götz.

  The microtubule-associated protein Tau is a driver of neuronal dysfunction in Alzheimer's disease and other tauopathies. In this process, Tau initially undergoes subtle changes to its abundance, subcellular localisation and a vast array of post-translational modifications including phosphorylation, that progressively result in the protein's somatodendritic accumulation and dysregulation of multiple Tau-dependent cellular processes. Given the various loss- and gain-of-functions of Tau in disease and the brain-wide changes in the proteome that characterise tauopathies, we asked whether targeting Tau would restore the alterations in proteostasis observed in disease. Therefore, by phage display, we generated a novel pan-Tau antibody, RNJ1, that preferentially binds human Tau and neutralises proteopathic seeding activity in multiple cell lines, and benchmarked it against a clinically tested pan-Tau antibody, HJ8.5 (murine version of tilavonemab). We then evaluated both antibodies, alone and in combination, in the K3 tauopathy mouse model, showing reduced Tau pathology and improvements in neuronal function following 14 weekly treatments, without obtaining synergy for the combination. These effects were more pronounced in female mice. To investigate the molecular mechanisms contributing to improvements in neuronal function, we employed quantitative proteomics, phosphoproteomics and kinase prediction analysis to first establish alterations in K3 mice relative to WT controls at the proteome level. In female K3 mice, we found 342 differentially abundant proteins, which are predominantly involved in metabolic and microtubule-associated processes, strengthening previously reported findings of defects in several functional domains in multiple tauopathy models. We next asked whether antibody-mediated Tau target engagement indirectly affects levels of deregulated proteins in the K3 model. Importantly, both immunotherapies, in particular RNJ1, induced abundance shifts towards a restoration to wild-type levels (proteostasis). A total of 257 of 342 (∼75%) proteins altered in K3 were closer in abundance to WT levels after RNJ1 treatment, and 73% after HJ8.5 treatment. However, the magnitude of these changes was less pronounced than that observed with RNJ1, as reflected by a far smaller number of differentially abundant proteins. Furthermore, analysis of the phosphoproteome showed an even stronger restoration effect with RNJ1, with ∼82% of altered phosphopeptides in K3 showing a shift to WT levels, and 75% with HJ8.5. Gene set over-representation analysis (ORA) further confirmed that proteins undergoing restoration are involved in biological pathways affected in K3 mice. Together, our study suggests that a Tau immunotherapy-induced restoration of proteostasis links target engagement and treatment efficacy.

Keywords:  Alzheimer’s disease; Tau antibodies; brain phosphoproteomics; brain proteomics; neurodegenerative diseases; quantitative mass spectrometry

DOI:  https://doi.org/10.1093/brain/awae254
Sci Adv. 2024 Aug 02. 10(31): eado9959

Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Ilana B Kotliar, Annika Bendes, Leo Dahl, Yuanhuang Chen, Marcus Saarinen, Emilie Ceraudo, Tea Dodig-Crnković, Mathias Uhlén, Per Svenningsson, Jochen M Schwenk, Thomas P Sakmar.

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

DOI: https://doi.org/10.1126/sciadv.ado9959
bioRxiv. 2024 Jul 16. pii: 2024.05.17.594758. [Epub ahead of print]

Massively parallel free energy calculations for in silico affinity maturation of designed miniproteins.

Dylan Novack, Si Zhang, Vincent A Voelz.

Computational protein design efforts continue to make remarkable advances, yet the discovery of high-affinity binders typically requires large-scale experimental screening of site-saturated mutant (SSM) libraries. Here, we explore how massively parallel free energy methods can be used for in silico affinity maturation of de novo designed binding proteins. Using an expanded ensemble (EE) approach, we perform exhaustive relative binding free energy calculations for SSM variants of three miniproteins designed to bind influenza A H1 hemagglutinin by Chevalier et al. (2017). We compare our predictions to experimental ΔΔ G values inferred from a Bayesian analysis of the high-throughput sequencing data, and to state-of-the-art predictions made using the Flex ddG Rosetta protocol. A systematic comparison reveals prediction accuracies around 2 kcal/mol, and identifies net charge changes, large numbers of alchemical atoms, and slow side chain conformational dynamics as key contributors to the uncertainty of the EE predictions. Flex ddG predictions are more accurate on average, but highly conservative. In contrast, EE predictions can better classify stabilizing and destabilizing mutations. We also explored the ability of SSM scans to rationalize known affinity-matured variants containing multiple mutations, which are non-additive due to epistatic effects. Simple electrostatic models fail to explain non-additivity, but observed mutations are found at positions with higher Shannon entropies. Overall, this work suggests that simulation-based free energy methods can provide predictive information for in silico affinity maturation of designed miniproteins, with many feasible improvements to the efficiency and accuracy within reach.

DOI: https://doi.org/10.1101/2024.05.17.594758
Nat Commun. 2024 Aug 01. 15(1): 6477

Dual-site molecular glues for enhancing protein-protein interactions of the CDK12-DDB1 complex.

Zemin Zhang, Yuanqing Li, Jie Yang, Jiacheng Li, Xiongqiang Lin, Ting Liu, Shiling Yang, Jin Lin, Shengyu Xue, Jiamin Yu, Cailing Tang, Ziteng Li, Liping Liu, Zhengzheng Ye, Yanan Deng, Zhihai Li, Kaixian Chen, Hong Ding, Cheng Luo, Hua Lin.

Protein-protein interactions (PPIs) stabilization with molecular glues plays a crucial role in drug discovery, albeit with significant challenges. In this study, we propose a dual-site approach, targeting the PPI region and its dynamic surroundings. We conduct molecular dynamics simulations to identify critical sites on the PPI that stabilize the cyclin-dependent kinase 12 - DNA damage-binding protein 1 (CDK12-DDB1) complex, resulting in further cyclin K degradation. This exploration leads to the creation of LL-K12-18, a dual-site molecular glue, which enhances the glue properties to augment degradation kinetics and efficiency. Notably, LL-K12-18 demonstrates strong inhibition of gene transcription and anti-proliferative effects in tumor cells, showing significant potency improvements in MDA-MB-231 (88-fold) and MDA-MB-468 cells (307-fold) when compared to its precursor compound SR-4835. These findings underscore the potential of dual-site approaches in disrupting CDK12 function and offer a structural insight-based framework for the design of cyclin K molecular glues.

DOI: https://doi.org/10.1038/s41467-024-50642-0
Nat Med. 2024 Aug 02.

Targeting axonal guidance dependencies in glioblastoma with ROBO1 CAR T cells.

Chirayu R Chokshi, Muhammad Vaseem Shaikh, Benjamin Brakel, Martin A Rossotti, David Tieu, William Maich, Alisha Anand, Shawn C Chafe, Kui Zhai, Yujin Suk, Agata M Kieliszek, Petar Miletic, Nicholas Mikolajewicz, David Chen, Jamie D McNicol, Katherine Chan, Amy H Y Tong, Laura Kuhlmann, Lina Liu, Zahra Alizada, Daniel Mobilio, Nazanin Tatari, Neil Savage, Nikoo Aghaei, Shan Grewal, Anish Puri, Minomi Subapanditha, Dillon McKenna, Vladimir Ignatchenko, Joseph M Salamoun, Jacek M Kwiecien, Peter Wipf, Elizabeth R Sharlow, John P Provias, Jian-Qiang Lu, John S Lazo, Thomas Kislinger, Yu Lu, Kevin R Brown, Chitra Venugopal, Kevin A Henry, Jason Moffat, Sheila K Singh.

Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. In this study, we investigated functional drivers of post-treatment recurrent GBM through integrative genomic analyses, genome-wide genetic perturbation screens in patient-derived GBM models and independent lines of validation. Specific genetic dependencies were found consistent across recurrent tumor models, accompanied by increased mutational burden and differential transcript and protein expression compared to its primary GBM predecessor. Our observations suggest a multi-layered genetic response to drive tumor recurrence and implicate PTP4A2 (protein tyrosine phosphatase 4A2) as a modulator of self-renewal, proliferation and tumorigenicity in recurrent GBM. Genetic perturbation or small-molecule inhibition of PTP4A2 acts through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1) and its downstream molecular players, exploiting a functional dependency on ROBO signaling. Because a pan-PTP4A inhibitor was limited by poor penetrance across the blood-brain barrier in vivo, we engineered a second-generation chimeric antigen receptor (CAR) T cell therapy against ROBO1, a cell surface receptor enriched across recurrent GBM specimens. A single dose of ROBO1-targeted CAR T cells doubled median survival in cell-line-derived xenograft (CDX) models of recurrent GBM. Moreover, in CDX models of adult lung-to-brain metastases and pediatric relapsed medulloblastoma, ROBO1 CAR T cells eradicated tumors in 50-100% of mice. Our study identifies a promising multi-targetable PTP4A-ROBO1 signaling axis that drives tumorigenicity in recurrent GBM, with potential in other malignant brain tumors.

DOI: https://doi.org/10.1038/s41591-024-03138-9
Neural Regen Res. 2025 May 01. 20(5): 1455-1466

Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury: PERK as a potential target for intervention.

Ju Zheng, Yixin Li, Ting Zhang, Yanlin Fu, Peiyan Long, Xiao Gao, Zhengwei Wang, Zhizhong Guan, Xiaolan Qi, Wei Hong, Yan Xiao.

JOURNAL/nrgr/04.03/01300535-202505000-00028/figure1/v/2024-07-28T173839Z/r/image-tiff Several studies have shown that activation of unfolded protein response and endoplasmic reticulum (ER) stress plays a crucial role in severe cerebral ischemia/reperfusion injury. Autophagy occurs within hours after cerebral ischemia, but the relationship between ER stress and autophagy remains unclear. In this study, we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury. We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2 subunit alpha (eIF2α)-activating transcription factor 4 (ATF4)-C/EBP homologous protein (CHOP), increased neuronal apoptosis, and induced autophagy. Furthermore, inhibition of ER stress using inhibitors or by siRNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis, indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy. Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis, indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury. Findings from this study indicate that cerebral ischemia/reperfusion injury can trigger neuronal ER stress and promote autophagy, and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

DOI: https://doi.org/10.4103/NRR.NRR-D-23-00794
Elife. 2024 Aug 01. pii: RP94755. [Epub ahead of print]13

Impact of protein and small molecule interactions on kinase conformations.

Valentina Kugler, Selina Schwaighofer, Andreas Feichtner, Florian Enzler, Jakob Fleischmann, Sophie Strich, Sarah Schwarz, Rebecca Wilson, Philipp Tschaikner, Jakob Troppmair, Veronika Sexl, Pascal Meier, Teresa Kaserer, Eduard Stefan.

  Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Keywords:  biochemistry; cell-based reporter assay; chemical biology; dark kinome; drug interactions; enzyme reactivation; hard-to-target enzymes; kinase complex formation; kinase conformation; kinase scaffolding function; none; pseudo enzymes; target engagement

DOI:  https://doi.org/10.7554/eLife.94755
Nat Commun. 2024 Jul 31. 15(1): 6469

The influence of HLA genetic variation on plasma protein expression.

Chirag Krishna, Joshua Chiou, Saori Sakaue, Joyce B Kang, Stephen M Christensen, Isac Lee, Melis Atalar Aksit, Hye In Kim, David von Schack, Soumya Raychaudhuri, Daniel Ziemek, Xinli Hu.

Genetic variation in the human leukocyte antigen (HLA) loci is associated with risk of immune-mediated diseases, but the molecular effects of HLA polymorphism are unclear. Here we examined the effects of HLA genetic variation on the expression of 2940 plasma proteins across 45,330 Europeans in the UK Biobank, with replication analyses across multiple ancestry groups. We detected 504 proteins affected by HLA variants (HLA-pQTL), including widespread trans effects by autoimmune disease risk alleles. More than 80% of the HLA-pQTL fine-mapped to amino acid positions in the peptide binding groove. HLA-I and II affected proteins expressed in similar cell types but in different pathways of both adaptive and innate immunity. Finally, we investigated potential HLA-pQTL effects on disease by integrating HLA-pQTL with fine-mapped HLA-disease signals in the UK Biobank. Our data reveal the diverse effects of HLA genetic variation and aid the interpretation of associations between HLA alleles and immune-mediated diseases.

DOI: https://doi.org/10.1038/s41467-024-50583-8