bims-proteo 2023-11-19 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–11–19
thirty papers selected by
Eric Chevet, INSERM

Elucidation of E3 ubiquitin ligase specificity through proteome-wide internal degron mapping.
RNF14-dependent atypical ubiquitylation promotes translation-coupled resolution of RNA-protein crosslinks.
EMC rectifies the topology of multipass membrane proteins.
Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.
PDZD8 promotes autophagy at ER-Lysosome contact sites to regulate synaptogenesis.
K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.
The E3 ligase Riplet promotes RIG-I signaling independent of RIG-I oligomerization.
LARP1 senses free ribosomes to coordinate supply and demand of ribosomal proteins.
A Critical Discussion on the relationship between E3 ubiquitin ligases, Protein Degradation and Skeletal Muscle Wasting: It's not that simple.
Mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN.
MAPL loss dysregulates bile and liver metabolism in mice.
The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation.
The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.
Uncovering DUB Selectivity through an Ion Mobility-Based Assessment of Ubiquitin Chain Isomers.
An adaptive stress response that confers cellular resilience to decreased ubiquitination.
Local membrane source gathering by p62 body drives autophagosome formation.
Mitochondria-associated membrane collapse impairs TBK1-mediated proteostatic stress response in ALS.
Recruitment of FBXO22 for Targeted Degradation of NSD2.
Platelet proteo-transcriptomic profiling validates mediators of thrombosis and proteostasis in patients with myeloproliferative neoplasms.
Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.
Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.
The essential chaperone DNAJC17 activates HSP70 to coordinate RNA splicing and G2-M progression.
Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins.
Stress granules plug and stabilize damaged endolysosomal membranes.
Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at endoplasmic reticulum monitoring and regulating fatty acids.
Hsp90 shapes adaptation by controlling the fitness consequences of regulatory variation.
The FERONIA-YUELAO module participates in translational control by modulating the abundance of tRNA fragments in Arabidopsis.
The social and structural architecture of the yeast protein interactome.
Genome mining yields putative disease-associated ROMK variants with distinct defects.
HSP47 levels determine the degree of body adiposity.

Mol Cell. 2023 Nov 16. pii: S1097-2765(23)00858-4. [Epub ahead of print]83(22): 4191-4192

Elucidation of E3 ubiquitin ligase specificity through proteome-wide internal degron mapping.

Zhiqian Zhang, Brandon Sie, Aiquan Chang, Yumei Leng, Christopher Nardone, Richard T Timms, Stephen J Elledge.

DOI: https://doi.org/10.1016/j.molcel.2023.10.021
Mol Cell. 2023 Nov 02. pii: S1097-2765(23)00849-3. [Epub ahead of print]

RNF14-dependent atypical ubiquitylation promotes translation-coupled resolution of RNA-protein crosslinks.

Shubo Zhao, Jacqueline Cordes, Karolina M Caban, Maximilian J Götz, Timur Mackens-Kiani, Anthony J Veltri, Niladri K Sinha, Pedro Weickert, Selay Kaya, Graeme Hewitt, Danny D Nedialkova, Thomas Fröhlich, Roland Beckmann, Allen R Buskirk, Rachel Green, Julian Stingele.

  Reactive aldehydes are abundant endogenous metabolites that challenge homeostasis by crosslinking cellular macromolecules. Aldehyde-induced DNA damage requires repair to prevent cancer and premature aging, but it is unknown whether cells also possess mechanisms that resolve aldehyde-induced RNA lesions. Here, we establish photoactivatable ribonucleoside-enhanced crosslinking (PAR-CL) as a model system to study RNA crosslinking damage in the absence of confounding DNA damage in human cells. We find that such RNA damage causes translation stress by stalling elongating ribosomes, which leads to collisions with trailing ribosomes and activation of multiple stress response pathways. Moreover, we discovered a translation-coupled quality control mechanism that resolves covalent RNA-protein crosslinks. Collisions between translating ribosomes and crosslinked mRNA-binding proteins trigger their modification with atypical K6- and K48-linked ubiquitin chains. Ubiquitylation requires the E3 ligase RNF14 and leads to proteasomal degradation of the protein adduct. Our findings identify RNA lesion-induced translational stress as a central component of crosslinking damage.

Keywords:  GCN1; K6-linked ubiquitin chains; RNA damage; RNA-protein crosslinks; RNF14; RNF25; atypical ubiquitylation; formaldehyde; ribosome; translation

DOI:  https://doi.org/10.1016/j.molcel.2023.10.012
Nat Struct Mol Biol. 2023 Nov 13.

EMC rectifies the topology of multipass membrane proteins.

Haoxi Wu, Luka Smalinskaitė, Ramanujan S Hegde.

Most eukaryotic multipass membrane proteins are inserted into the membrane of the endoplasmic reticulum. Their transmembrane domains (TMDs) are thought to be inserted co-translationally as they emerge from a membrane-bound ribosome. Here we find that TMDs near the carboxyl terminus of mammalian multipass proteins are inserted post-translationally by the endoplasmic reticulum membrane protein complex (EMC). Site-specific crosslinking shows that the EMC's cytosol-facing hydrophilic vestibule is adjacent to a pre-translocated C-terminal tail. EMC-mediated insertion is mostly agnostic to TMD hydrophobicity, favored for short uncharged C-tails and stimulated by a preceding unassembled TMD bundle. Thus, multipass membrane proteins can be released by the ribosome-translocon complex in an incompletely inserted state, requiring a separate EMC-mediated post-translational insertion step to rectify their topology, complete biogenesis and evade quality control. This sequential co-translational and post-translational mechanism may apply to ~250 diverse multipass proteins, including subunits of the pentameric ion channel family that are crucial for neurotransmission.

DOI: https://doi.org/10.1038/s41594-023-01120-6
Nat Struct Mol Biol. 2023 Nov;30(11): 1816-1825

Start codon-associated ribosomal frameshifting mediates nutrient stress adaptation.

Yuanhui Mao, Longfei Jia, Leiming Dong, Xin Erica Shu, Shu-Bing Qian.

A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. Start codon-associated ribosomal frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra that are unannotated in the human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational 'noise' in nutrient stress adaptation.

DOI: https://doi.org/10.1038/s41594-023-01119-z
bioRxiv. 2023 Oct 30. pii: 2023.10.30.564828. [Epub ahead of print]

PDZD8 promotes autophagy at ER-Lysosome contact sites to regulate synaptogenesis.

Rajan S Thakur, Kate M O'Connor-Giles.

Building synaptic connections, which are often far from the soma, requires coordinating a host of cellular activities from transcription to protein turnover, placing a high demand on intracellular communication. Membrane contact sites (MCSs) formed between cellular organelles have emerged as key signaling hubs for coordinating an array of cellular activities. We have found that the endoplasmic reticulum (ER) MCS tethering protein PDZD8 is required for activity-dependent synaptogenesis. PDZD8 is sufficient to drive ectopic synaptic bouton formation through an autophagy-dependent mechanism and required for basal synapse formation when autophagy biogenesis is limited. PDZD8 functions at ER-late endosome/lysosome (LEL) MCSs to promote lysosome maturation and accelerate autophagic flux. Mutational analysis of PDZD8's SMP domain further suggests a role for lipid transfer at ER-LEL MCSs. We propose that PDZD8-dependent lipid transfer from ER to LELs promotes lysosome maturation to increase autophagic flux during periods of high demand, including activity-dependent synapse formation.
GRAPHICAL ABSTRACT:

DOI: https://doi.org/10.1101/2023.10.30.564828
Mol Cell. 2023 Nov 02. pii: S1097-2765(23)00848-1. [Epub ahead of print]

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution.

Aldwin Suryo Rahmanto, Christian J Blum, Claudia Scalera, Jan B Heidelberger, Mikhail Mesitov, Daniel Horn-Ghetko, Justus F Gräf, Ivan Mikicic, Rebecca Hobrecht, Anna Orekhova, Matthias Ostermaier, Stefanie Ebersberger, Martin M Möckel, Nils Krapoth, Nádia Da Silva Fernandes, Athanasia Mizi, Yajie Zhu, Jia-Xuan Chen, Chunaram Choudhary, Argyris Papantonis, Helle D Ulrich, Brenda A Schulman, Julian König, Petra Beli.

  Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

Keywords:  K6-linked ubiquitylation; RNA-protein crosslinks; RNF14; VCP; quantitative proteomics; reactive aldehydes; ribosome; translation

DOI:  https://doi.org/10.1016/j.molcel.2023.10.011
Nat Commun. 2023 Nov 11. 14(1): 7308

The E3 ligase Riplet promotes RIG-I signaling independent of RIG-I oligomerization.

Wenshuai Wang, Benjamin Götte, Rong Guo, Anna Marie Pyle.

RIG-I is an essential innate immune receptor that responds to infection by RNA viruses. The RIG-I signaling cascade is mediated by a series of post-translational modifications, the most important of which is ubiquitination of the RIG-I Caspase Recruitment Domains (CARDs) by E3 ligase Riplet. This is required for interaction between RIG-I and its downstream adapter protein MAVS, but the mechanism of action remains unclear. Here we show that Riplet is required for RIG-I signaling in the presence of both short and long dsRNAs, establishing that Riplet activation does not depend upon RIG-I filament formation on long dsRNAs. Likewise, quantitative Riplet-RIG-I affinity measurements establish that Riplet interacts with RIG-I regardless of whether the receptor is bound to RNA. To understand this, we solved high-resolution cryo-EM structures of RIG-I/RNA/Riplet complexes, revealing molecular interfaces that control Riplet-mediated activation and enabling the formulation of a unified model for the role of Riplet in signaling.

DOI: https://doi.org/10.1038/s41467-023-42982-0
bioRxiv. 2023 Nov 02. pii: 2023.11.01.565189. [Epub ahead of print]

LARP1 senses free ribosomes to coordinate supply and demand of ribosomal proteins.

James A Saba, Zixuan Huang, Kate L Schole, Xianwen Ye, Shrey D Bhatt, Yi Li, Winston Timp, Jingdong Cheng, Rachel Green.

Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies implicate LARP1 in 40S- or 80S-ribosome complexes that repress and stabilize TOPs. However, a mechanistic understanding of how LARP1 and TOPs interact with these complexes to coordinate TOP outcomes is lacking. Here, we show that LARP1 senses the cellular supply of ribosomes by directly binding non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the 40S mRNA channel. Free cytosolic ribosomes induce sequestration of TOPs in repressed 80S-LARP1-TOP complexes independent of alterations in mTOR signaling. Together, this work demonstrates a general ribosome-sensing function of LARP1 that allows it to tune ribosome protein synthesis to cellular demand.
One-Sentence Summary: LARP1 directly binds free ribosomal subunits to repress TOP mRNAs.

DOI: https://doi.org/10.1101/2023.11.01.565189
Am J Physiol Cell Physiol. 2023 Nov 13.

A Critical Discussion on the relationship between E3 ubiquitin ligases, Protein Degradation and Skeletal Muscle Wasting: It's not that simple.

David C Hughes, Craig A Goodman, Leslie M Baehr, Paul Gregorevic, Sue C Bodine.

  Ubiquitination is an important post-translational modification for protein substrates, whereby ubiquitin is added to proteins through the coordinated activity of activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. The E3s provide key functions in the recognition of specific protein substrates to be ubiquitinated and aid in determining their proteolytic or non-proteolytic fates, which has led to their study as indicators of altered cellular processes. MuRF1 and MAFbx/Atrogin-1 were two of the first E3 ubiquitin ligases identified as being upregulated in a range of different skeletal muscle atrophy models. Since their discovery, the expression of these E3 ubiquitin ligases has often been studied as a surrogate measure of changes to bulk protein degradation rates. However, emerging evidence has highlighted the dynamic and complex regulation of the ubiquitin proteasome system in skeletal muscle and demonstrated that protein ubiquitination is not necessarily equivalent to protein degradation. These observations highlight the potential challenges of quantifying E3 ubiquitin ligases as markers of protein degradation rates or ubiquitin proteasome system (UPS) activation. This perspective examines the usefulness of monitoring E3 ubiquitin ligases for determining specific or bulk protein degradation rates in settings of skeletal muscle atrophy. Specific questions that remain unanswered within the skeletal muscle atrophy field are also identified, to encourage the pursuit of new research that will be critical in moving forward our understanding of the molecular mechanisms that govern protein function and degradation in muscle.

Keywords:  Atrophy; disuse; proteasome system; protein degradation; ubiquitination

DOI:  https://doi.org/10.1152/ajpcell.00457.2023
Biochem Biophys Res Commun. 2023 Nov 11. pii: S0006-291X(23)01333-5. [Epub ahead of print]689 149239

Mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN.

Yan Zhang, Xiaolong Xu, Yaru Wang, Yingli Wang, Xindi Zhou, Lifeng Pan.

  HOIL-1L and SHARPIN are two essential regulatory subunits of the linear ubiquitin chain assembly complex (LUBAC), which is the only known E3 ligase complex generating linear ubiquitin chains. In addition to their LUBAC-dependent functions, HOIL-1L and SHARPIN alone play crucial roles in many LUBAC-independent cellular processes. Importantly, deficiency of HOIL-1L or SHARPIN leads to severe disorders in humans or mice. However, the mechanistic bases underlying the multi-functions of HOIL-1L and SHARPIN are still largely unknown. Here, we uncover that HOIL-1L and SHARPIN alone can form homo-dimers through their LTM motifs. We solve two crystal structures of the dimeric LTM motifs of HOIL-1L and SHARPIN, which not only elucidate the detailed molecular mechanism underpinning the dimer formations of HOIL-1L and SHARPIN, but also reveal a general mode shared by the LTM motifs of HOIL-1L and SHARPIN for forming homo-dimer or hetero-dimer. Furthermore, we elucidate that the polyglucosan body myopathy-associated HOIL-1L A18P mutation disturbs the structural folding of HOIL-1L LTM, and disrupts the dimer formation of HOIL-1L. In summary, our study provides mechanistic insights into the homo-dimerization of HOIL-1L and SHARPIN mediated by their LTM motifs, and expands our understandings of the multi-functions of HOIL-1L and SHARPIN as well as the etiology of relevant human disease caused by defective HOIL-1L.

Keywords:  HOIL-1L; LTM motif; LUBAC; Polyglucosan body myopathy; SHARPIN

DOI:  https://doi.org/10.1016/j.bbrc.2023.149239
EMBO Rep. 2023 Nov 14. e57972

MAPL loss dysregulates bile and liver metabolism in mice.

Vanessa Goyon, Aurèle Besse-Patin, Rodolfo Zunino, Olesia Ignatenko, Mai Nguyen, Étienne Coyaud, Jonathan M Lee, Bich N Nguyen, Brian Raught, Heidi M McBride.

  Mitochondrial and peroxisomal anchored protein ligase (MAPL) is a dual ubiquitin and small ubiquitin-like modifier (SUMO) ligase with roles in mitochondrial quality control, cell death and inflammation in cultured cells. Here, we show that MAPL function in the organismal context converges on metabolic control, as knockout mice are viable, insulin-sensitive, and protected from diet-induced obesity. MAPL loss leads to liver-specific activation of the integrated stress response, inducing secretion of stress hormone FGF21. MAPL knockout mice develop fully penetrant spontaneous hepatocellular carcinoma. Mechanistically, the peroxisomal bile acid transporter ABCD3 is a primary MAPL interacting partner and SUMOylated in a MAPL-dependent manner. MAPL knockout leads to increased bile acid production coupled with defective regulatory feedback in liver in vivo and in isolated primary hepatocytes, suggesting cell-autonomous function. Together, our findings establish MAPL function as a regulator of bile acid synthesis whose loss leads to the disruption of bile acid feedback mechanisms. The consequences of MAPL loss in liver, along with evidence of tumor suppression through regulation of cell survival pathways, ultimately lead to hepatocellular carcinogenesis.

Keywords:  MUL1; PMP70; SUMO; hepatocellular carcinoma; peroxisome

DOI:  https://doi.org/10.15252/embr.202357972
bioRxiv. 2023 Oct 23. pii: 2023.10.23.563604. [Epub ahead of print]

The Ribosome Assembly Factor Reh1 is Released from the Polypeptide Exit Tunnel in the Pioneer Round of Translation.

Sharmishtha Musalgaonkar, James Yelland, Ruta Chitale, Shilpa Rao, Hakan Ozadam, Can Cenik, David Taylor, Arlen Johnson.

Assembly of functional ribosomal subunits and successfully delivering them to the translating pool is a prerequisite for protein synthesis and cell growth. In S. cerevisiae, the ribosome assembly factor Reh1 binds to pre-60S subunits at a late stage during their cytoplasmic maturation. Previous work shows that the C-terminus of Reh1 inserts into the polypeptide exit tunnel (PET) of the pre-60S subunit. Unlike canonical assembly factors, which associate exclusively with pre-60S subunits, we observed that Reh1 sediments with polysomes in addition to free 60S subunits. We therefore investigated the intriguing possibility that Reh1 remains associated with 60S subunits after the release of the anti-association factor Tif6 and after subunit joining. Here, we show that Reh1-bound nascent 60S subunits associate with 40S subunits to form actively translating ribosomes. Using selective ribosome profiling, we found that Reh1-bound ribosomes populate open reading frames near start codons. Reh1-bound ribosomes are also strongly enriched for initiator tRNA, indicating they are associated with early elongation events. Using single particle cryo-electron microscopy to image cycloheximide-arrested Reh1-bound 80S ribosomes, we found that Reh1-bound 80S contain A site peptidyl tRNA, P site tRNA and eIF5A indicating that Reh1 does not dissociate from 60S until early stages of translation elongation. We propose that Reh1 is displaced by the elongating peptide chain. These results identify Reh1 as the last assembly factor released from the nascent 60S subunit during its pioneer round of translation.

DOI: https://doi.org/10.1101/2023.10.23.563604
bioRxiv. 2023 Nov 04. pii: 2023.11.03.565564. [Epub ahead of print]

The membrane-associated ubiquitin ligase MARCHF8 stabilizes the human papillomavirus oncoprotein E7 by degrading CUL1 and UBE2L3 in head and neck cancer.

Mohamed I Khalil, Canchai Yang, Lexi Vu, Smriti Chadha, Harrison Nabors, Claire D James, Iain M Morgan, Dohun Pyeon.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the E7 protein by degrading the components of the SKP1-CUL1-F-box (SCF) ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases E7 protein levels and suppresses tumor growth in vivo. Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.
IMPORTANCE: Since HPV oncoprotein E7 is essential for virus replication, HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the components of the E3 ubiquitin ligase complex CUL1 and UBE2L3 play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase MARCHF8 induced by HPV E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis.

DOI: https://doi.org/10.1101/2023.11.03.565564
Anal Chem. 2023 Nov 14.

Uncovering DUB Selectivity through an Ion Mobility-Based Assessment of Ubiquitin Chain Isomers.

Elizaveta I Shestoperova, Eric R Strieter.

Ubiquitination is a reversible post-translational modification that maintains cellular homeostasis and regulates protein turnover. Deubiquitinases (DUBs) are a large family of proteases that catalyze the removal of ubiquitin (Ub) along with the dismantling and editing of Ub chains. Assessing the activity and selectivity of DUBs is critical for defining physiological functions. Despite numerous methods for evaluating DUB activity, none are capable of assessing activity and selectivity in the context of multicomponent mixtures of native unlabeled Ub conjugates. Here, we report an ion mobility (IM)-based approach for measuring DUB selectivity in the context of unlabeled mixtures of Ub chains. We show that IM-mass spectrometry (IM-MS) can be used to assess the selectivity of DUBs in a time-dependent manner. Moreover, using the branched Ub chain selective DUB UCH37/UCHL5 along with a mixture of Ub trimers, a strong preference for branched Ub trimers bearing K6 and K48 linkages is revealed. Our results demonstrate that IM-MS is a powerful method for evaluating DUB selectivity under conditions more physiologically relevant than single-component mixtures.

DOI: https://doi.org/10.1021/acs.analchem.3c04622
Nat Commun. 2023 Nov 14. 14(1): 7348

An adaptive stress response that confers cellular resilience to decreased ubiquitination.

Liam C Hunt, Vishwajeeth Pagala, Anna Stephan, Boer Xie, Kiran Kodali, Kanisha Kavdia, Yong-Dong Wang, Abbas Shirinifard, Michelle Curley, Flavia A Graca, Yingxue Fu, Suresh Poudel, Yuxin Li, Xusheng Wang, Haiyan Tan, Junmin Peng, Fabio Demontis.

Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.

DOI: https://doi.org/10.1038/s41467-023-43262-7
Nat Commun. 2023 Nov 13. 14(1): 7338

Local membrane source gathering by p62 body drives autophagosome formation.

Xuezhao Feng, Daxiao Sun, Yanchang Li, Jinpei Zhang, Shiyu Liu, Dachuan Zhang, Jingxiang Zheng, Qing Xi, Haisha Liang, Wenkang Zhao, Ying Li, Mengbo Xu, Jiayu He, Tong Liu, Ayshamgul Hasim, Meisheng Ma, Ping Xu, Na Mi.

Autophagosomes are double-membrane vesicles generated intracellularly to encapsulate substrates for lysosomal degradation during autophagy. Phase separated p62 body plays pivotal roles during autophagosome formation, however, the underlying mechanisms are still not fully understood. Here we describe a spatial membrane gathering mode by which p62 body functions in autophagosome formation. Mass spectrometry-based proteomics reveals significant enrichment of vesicle trafficking components within p62 body. Combining cellular experiments and biochemical reconstitution assays, we confirm the gathering of ATG9 and ATG16L1-positive vesicles around p62 body, especially in Atg2ab DKO cells with blocked lipid transfer and vesicle fusion. Interestingly, p62 body also regulates ATG9 and ATG16L vesicle trafficking flux intracellularly. We further determine the lipid contents associated with p62 body via lipidomic profiling. Moreover, with in vitro kinase assay, we uncover the functions of p62 body as a platform to assemble ULK1 complex and invigorate PI3KC3-C1 kinase cascade for PI3P generation. Collectively, our study raises a membrane-based working model for multifaceted p62 body in controlling autophagosome biogenesis, and highlights the interplay between membraneless condensates and membrane vesicles in regulating cellular functions.

DOI: https://doi.org/10.1038/s41467-023-42829-8
Proc Natl Acad Sci U S A. 2023 Nov 21. 120(47): e2315347120

Mitochondria-associated membrane collapse impairs TBK1-mediated proteostatic stress response in ALS.

Seiji Watanabe, Yuri Murata, Yasuyoshi Oka, Kotaro Oiwa, Mai Horiuchi, Yohei Iguchi, Okiru Komine, Akira Sobue, Masahisa Katsuno, Tomoo Ogi, Koji Yamanaka.

  The organelle contact site of the endoplasmic reticulum and mitochondria, known as the mitochondria-associated membrane (MAM), is a multifunctional microdomain in cellular homeostasis. We previously reported that MAM disruption is a common pathological feature in amyotrophic lateral sclerosis (ALS); however, the precise role of MAM in ALS was uncovered. Here, we show that the MAM is essential for TANK-binding kinase 1 (TBK1) activation under proteostatic stress conditions. A MAM-specific E3 ubiquitin ligase, autocrine motility factor receptor, ubiquitinated nascent proteins to activate TBK1 at the MAM, which results in ribosomal protein degradation. MAM or TBK1 deficiency under proteostatic stress conditions resulted in increased cellular vulnerability in vitro and motor impairment in vivo. Thus, MAM disruption exacerbates proteostatic stress via TBK1 inactivation in ALS. Our study has revealed a proteostatic mechanism mediated by the MAM-TBK1 axis, highlighting the physiological importance of the organelle contact sites.

Keywords:  TANK-binding kinase 1; amyotrophic lateral sclerosis; mitochondria-associated membrane; sigma-1 receptor; stress granules

DOI:  https://doi.org/10.1073/pnas.2315347120
bioRxiv. 2023 Nov 02. pii: 2023.11.01.564830. [Epub ahead of print]

Recruitment of FBXO22 for Targeted Degradation of NSD2.

David Y Nie, John R Tabor, Jianping Li, Maria Kutera, Jonathan St-Germain, Ronan P Hanley, Esther Wolf, Ethan Paulakonis, Tristan M G Kenney, Shili Duan, Suman Shrestha, Dominic D G Owens, Ailing Pon, Magdalena Szewczyk, Anthony Joseph Lamberto, Michael Menes, Fengling Li, Dalia Barsyte-Lovejoy, Nicholas G Brown, Anthony M Barsotti, Andrew W Stamford, Jon L Collins, Derek J Wilson, Brian Raught, Jonathan D Licht, Lindsey I James, Cheryl H Arrowsmith.

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

DOI: https://doi.org/10.1101/2023.11.01.564830
bioRxiv. 2023 Oct 26. pii: 2023.10.23.563619. [Epub ahead of print]

Platelet proteo-transcriptomic profiling validates mediators of thrombosis and proteostasis in patients with myeloproliferative neoplasms.

Sarah Kelliher, Sara Gamba, Luisa Weiss, Zhu Shen, Marina Marchetti, Francesca Schieppati, Caitriona Scaife, Stephen Madden, Kathleen Bennett, Anne Fortune, Su Maung, Michael Fay, Fionnuala Ní Áinle, Patricia Maguire, Anna Falanga, Barry Kevane, Anandi Krishnan.

Patients with chronic Myeloproliferative Neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia (ET) exhibit unique clinical features, such as a tendency toward thrombosis and hemorrhage, and risk of disease progression to secondary bone marrow fibrosis and/or acute leukemia. Although an increase in blood cell lineage counts (quantitative features) contribute to these morbid sequelae, the significant qualitative abnormalities of myeloid cells that contribute to vascular risk are not well understood. Here, we address this critical knowledge gap via a comprehensive and untargeted profiling of the platelet proteome in a large (n= 140) cohort of patients (from two independent sites) with an established diagnosis of PV and ET (and complement prior work on the MPN platelet transcriptome from a third site). We discover distinct MPN platelet protein expression and confirm key molecular impairments associated with proteostasis and thrombosis mechanisms of potential relevance to MPN pathology. Specifically, we validate expression of high-priority candidate markers from the platelet transcriptome at the platelet proteome ( e.g., calreticulin (CALR), Fc gamma receptor (FcγRIIA ) and galectin-1 (LGALS1 ) pointing to their likely significance in the proinflammatory, prothrombotic and profibrotic phenotypes in patients with MPN. Together, our proteo-transcriptomic study identifies the peripherally-derived platelet molecular profile as a potential window into MPN pathophysiology and demonstrates the value of integrative multi-omic approaches in gaining a better understanding of the complex molecular dynamics of disease.
Highlights: MPN patient platelet proteome identifies key pathobiological mediators of thrombosis and proteostasis. The MPN platelet proteomic profile validates our prior findings from the platelet transcriptome.

DOI: https://doi.org/10.1101/2023.10.23.563619
bioRxiv. 2023 Oct 26. pii: 2023.10.26.563984. [Epub ahead of print]

Mapping RNA-Protein Interactions with Subcellular Resolution Using Colocalization CLIP.

Soon Yi, Shashi S Singh, Kathryn Rozen-Gagnon, Joseph M Luna.

RNA binding proteins (RBPs) are essential for RNA metabolism and profoundly impact health and disease. The subcellular organization of RBP interaction networks with target RNAs remains largely unexplored. Here, we develop colocalization CLIP, a method that combines CrossLinking and ImmunoPrecipitation (CLIP) with proximity labeling, to explore in-depth the subcellular RNA interactions of the well-studied RNA-binding protein HuR. Using this method, we uncover HuR's dynamic and location-specific interactions with RNA, revealing alterations in sequence preferences and interactions in the nucleus, cytosol, or stress granule compartments. We uncover HuR's unique binding preferences within stress granules during arsenite stress, illuminating intricate interactions that conventional methodologies cannot capture. Overall, coCLIP provides a powerful method for revealing RBP:RNA interactions based on localization and lays the foundation for an advanced understanding of RBP models that incorporate subcellular location as a critical determinant of their functions.

DOI: https://doi.org/10.1101/2023.10.26.563984
Cell Chem Biol. 2023 Nov 06. pii: S2451-9456(23)00374-4. [Epub ahead of print]

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

Neetika Jaisinghani, Mary L Previti, Joshua Andrade, Manor Askenazi, Beatrix Ueberheide, Jessica C Seeliger.

  The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Keywords:  APEX2; ESX; Type VII secretion; cell wall; mycobacteria; periplasm; proximity labeling; tuberculosis

DOI:  https://doi.org/10.1016/j.chembiol.2023.10.013
bioRxiv. 2023 Oct 26. pii: 2023.10.25.564066. [Epub ahead of print]

The essential chaperone DNAJC17 activates HSP70 to coordinate RNA splicing and G2-M progression.

David V Allegakoen, Kristen Kwong, Jacqueline Morales, Trever G Bivona, Amit J Sabnis.

Molecular chaperones including the heat-shock protein 70-kilodalton (HSP70) family and the J-domain containing protein (JDP) co-chaperones maintain homeostatic balance in eukaryotic cells through regulation of the proteome. The expansive JDP family helps direct specific HSP70 functions, and yet loss of single JDP-encoding genes is widely tolerated by mammalian cells, suggesting a high degree of redundancy. By contrast, essential JDPs might carry out HSP70-independent functions or fill cell-context dependent, highly specialized roles within the proteostasis network. Using a genetic screen of JDPs in human cancer cell lines, we found the RNA recognition motif (RRM) containing DNAJC17 to be pan-essential and investigated the contribution of its structural domains to biochemical and cellular function. We found that the RRM exerts an auto-inhibitory effect on the ability of DNAJC17 to allosterically activate ATP hydrolysis by HSP70. The J-domain, but neither the RRM nor a distal C-terminal alpha helix are required to rescue cell viability after loss of endogenous DNAJC17 . Knockdown of DNAJC17 leads to relatively few conserved changes in the abundance of individual mRNAs, but instead deranges gene expression through exon skipping, primarily of genes involved in cell cycle progression. Concordant with cell viability experiments, the C-terminal portions of DNAJC17 are dispensable for restoring splicing and G2-M progression. Overall, our findings identify essential cellular JDPs and suggest that diversification in JDP structure extends the HSP70-JDP system to control divergent processes such as RNA splicing. Future investigations into the structural basis for auto-inhibition of the DNAJC17 J-domain and the molecular regulation of splicing by these components may provide insights on how conserved biochemical mechanisms can be programmed to fill unique, non-redundant cellular roles and broaden the scope of the proteostasis network.

DOI: https://doi.org/10.1101/2023.10.25.564066
bioRxiv. 2023 Oct 27. pii: 2023.10.26.564194. [Epub ahead of print]

Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins.

Changliang Chen, Mo Chen, Tianmu Wen, Richard A Anderson, Vincent L Cryns.

Reactive oxygen species (ROS) are generated by aerobic metabolism, and their deleterious effects are buffered by the cellular antioxidant response, which prevents oxidative stress. The nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of the antioxidant response. Basal levels of NRF2 are kept low by ubiquitin-dependent degradation of NRF2 by E3 ligases, including the Kelch-like ECH-associated protein 1 (KEAP1). Here, we show that the stability and function of NRF2 is regulated by the type I phosphatidylinositol phosphate kinase g (PIPKIg), which binds NRF2 and transfers its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) to NRF2. PtdIns(4,5)P 2 binding recruits the small heat shock protein HSP27 to the complex. Silencing PIPKIg or HSP27 destabilizes NRF2, reduces expression of its target gene HO-1, and sensitizes cells to oxidative stress. These data demonstrate an unexpected role of phosphoinositides and HSP27 in regulating NRF2 and point to PIPKIg and HSP27 as drug targets to destabilize NRF2 in cancer.
In brief: Phosphoinositides are coupled to NRF2 by PIPKIγ, and HSP27 is recruited and stabilizes NRF2, promoting stress-resistance.

DOI: https://doi.org/10.1101/2023.10.26.564194
Nature. 2023 Nov 15.

Stress granules plug and stabilize damaged endolysosomal membranes.

Claudio Bussi, Agustín Mangiarotti, Christian Vanhille-Campos, Beren Aylan, Enrica Pellegrino, Natalia Athanasiadi, Antony Fearns, Angela Rodgers, Titus M Franzmann, Anđela Šarić, Rumiana Dimova, Maximiliano G Gutierrez.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

DOI: https://doi.org/10.1038/s41586-023-06726-w
PNAS Nexus. 2023 Nov;2(11): pgad351

Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at endoplasmic reticulum monitoring and regulating fatty acids.

Song-Iee Han, Masanori Nakakuki, Yoshimi Nakagawa, Yunong Wang, Masaya Araki, Yuta Yamamoto, Hiroaki Tokiwa, Hiroyuki Takeda, Yuhei Mizunoe, Kaori Motomura, Hiroshi Ohno, Kenta Kainoh, Yuki Murayama, Yuichi Aita, Yoshinori Takeuchi, Yoshinori Osaki, Takafumi Miyamoto, Motohiro Sekiya, Takashi Matsuzaka, Naoya Yahagi, Hirohito Sone, Hiroaki Daitoku, Ryuichiro Sato, Hiroyuki Kawano, Hitoshi Shimano.

  The endoplasmic reticulum (ER)-embedded transcription factors, sterol regulatory element-binding proteins (SREBPs), master regulators of lipid biosynthesis, are transported to the Golgi for proteolytic activation to tune cellular cholesterol levels and regulate lipogenesis. However, mechanisms by which the cell responds to the levels of saturated or unsaturated fatty acids remain underexplored. Here, we show that RHBDL4/RHBDD1, a rhomboid family protease, directly cleaves SREBP-1c at the ER. The p97/VCP, AAA-ATPase complex then acts as an auxiliary segregase to extract the remaining ER-embedded fragment of SREBP-1c. Importantly, the enzymatic activity of RHBDL4 is enhanced by saturated fatty acids (SFAs) but inhibited by polyunsaturated fatty acids (PUFAs). Genetic deletion of RHBDL4 in mice fed on a Western diet enriched in SFAs and cholesterol prevented SREBP-1c from inducing genes for lipogenesis, particularly for synthesis and incorporation of PUFAs, and secretion of lipoproteins. The RHBDL4-SREBP-1c pathway reveals a regulatory system for monitoring fatty acid composition and maintaining cellular lipid homeostasis.

Keywords:  PUFA; RHBDL4; SREBP-1c; p97/VCP

DOI:  https://doi.org/10.1093/pnasnexus/pgad351
bioRxiv. 2023 Nov 02. pii: 2023.10.30.564848. [Epub ahead of print]

Hsp90 shapes adaptation by controlling the fitness consequences of regulatory variation.

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

The essential stress-responsive chaperone Hsp90 impacts development and adaptation from microbes to humans. Yet despite evidence of its role in evolution, pathogenesis, and oncogenic transformation, the molecular mechanisms by which Hsp90 alters the consequences of mutations remain vigorously debated. Here we exploit the power of nucleotide-resolution genetic mapping in Saccharomyces cerevisiae to uncover more than 1,000 natural variant-to-phenotype associations governed by this molecular chaperone. Strikingly, Hsp90 more frequently modified the phenotypic effects of cis -regulatory variation than variants that altered protein sequence. Moreover, these interactions made the largest contribution to Hsp90-dependent heredity. Nearly all interacting variants-both regulatory and protein-coding-fell within clients of Hsp90 or targets of its direct binding partners. Hsp90 activity affected mutations in evolutionarily young genes, segmental deletions, and heterozygotes, highlighting its influence on variation central to evolutionary novelty. Reconciling the diverse epistatic effects of this chaperone, synthetic transcriptional regulation and reconstructions of natural alleles by genome editing revealed a central role for Hsp90 in regulating the fundamental relationship between activity and phenotype. Our findings establish that non-coding variation is a core driver of Hsp90's influence on heredity, offering a mechanistic explanation for the chaperone's strong effects on evolution and development across species.

DOI: https://doi.org/10.1101/2023.10.30.564848
Dev Cell. 2023 Nov 09. pii: S1534-5807(23)00557-9. [Epub ahead of print]

The FERONIA-YUELAO module participates in translational control by modulating the abundance of tRNA fragments in Arabidopsis.

Sirui Zhu, Yuanyuan Li, You Wu, Yanan Shen, Ying Wang, Yujie Yan, Weijun Chen, Qiong Fu, Yirong Wang, Xiang Yu, Feng Yu.

  tRNA fragments (tRFs) are a recently identified class of small noncoding RNAs. To date, the regulation of tRF abundance and its functional mechanisms have been largely unclear in plants. We investigated how the Arabidopsis thaliana receptor kinase FERONIA (FER) regulates the abundance of tRFs to inhibit global mRNA translation. We demonstrate that FER regulates tRF abundance by directly phosphorylating the tRNA-binding protein YUELAO (YL) to modulate its function. Downregulation of FER and YL prevented the modification of tRNA via cytosine-5-methylation and 2'-O-methylation, thereby increasing tRF abundance. Furthermore, we show that YL acts as an important genetic downstream target of FER signaling, and knockdown of a specific tRF partially rescues the root hair growth defects of fer and yl mutants. Our findings shed light on the abundance and regulatory mechanisms of tRF and their role in inhibiting translation in plants.

Keywords:  RNA metabolism; YUELAO; protein biosynthesis; receptor kinase FER; tRF

DOI:  https://doi.org/10.1016/j.devcel.2023.10.014
Nature. 2023 Nov 15.

The social and structural architecture of the yeast protein interactome.

André C Michaelis, Andreas-David Brunner, Maximilian Zwiebel, Florian Meier, Maximilian T Strauss, Isabell Bludau, Matthias Mann.

Cellular functions are mediated by protein-protein interactions, and mapping the interactome provides fundamental insights into biological systems. Affinity purification coupled to mass spectrometry is an ideal tool for such mapping, but it has been difficult to identify low copy number complexes, membrane complexes and complexes that are disrupted by protein tagging. As a result, our current knowledge of the interactome is far from complete, and assessing the reliability of reported interactions is challenging. Here we develop a sensitive high-throughput method using highly reproducible affinity enrichment coupled to mass spectrometry combined with a quantitative two-dimensional analysis strategy to comprehensively map the interactome of Saccharomyces cerevisiae. Thousand-fold reduced volumes in 96-well format enabled replicate analysis of the endogenous GFP-tagged library covering the entire expressed yeast proteome1. The 4,159 pull-downs generated a highly structured network of 3,927 proteins connected by 31,004 interactions, doubling the number of proteins and tripling the number of reliable interactions compared with existing interactome maps2. This includes very-low-abundance epigenetic complexes, organellar membrane complexes and non-taggable complexes inferred by abundance correlation. This nearly saturated interactome reveals that the vast majority of yeast proteins are highly connected, with an average of 16 interactors. Similar to social networks between humans, the average shortest distance between proteins is 4.2 interactions. AlphaFold-Multimer provided novel insights into the functional roles of previously uncharacterized proteins in complexes. Our web portal ( www.yeast-interactome.org ) enables extensive exploration of the interactome dataset.

DOI: https://doi.org/10.1038/s41586-023-06739-5
PLoS Genet. 2023 Nov 13. 19(11): e1011051

Genome mining yields putative disease-associated ROMK variants with distinct defects.

Nga H Nguyen, Srikant Sarangi, Erin M McChesney, Shaohu Sheng, Jacob D Durrant, Aidan W Porter, Thomas R Kleyman, Zachary W Pitluk, Jeffrey L Brodsky.

Bartter syndrome is a group of rare genetic disorders that compromise kidney function by impairing electrolyte reabsorption. Left untreated, the resulting hyponatremia, hypokalemia, and dehydration can be fatal, and there is currently no cure. Bartter syndrome type II specifically arises from mutations in KCNJ1, which encodes the renal outer medullary potassium channel, ROMK. Over 40 Bartter syndrome-associated mutations in KCNJ1 have been identified, yet their molecular defects are mostly uncharacterized. Nevertheless, a subset of disease-linked mutations compromise ROMK folding in the endoplasmic reticulum (ER), which in turn results in premature degradation via the ER associated degradation (ERAD) pathway. To identify uncharacterized human variants that might similarly lead to premature degradation and thus disease, we mined three genomic databases. First, phenotypic data in the UK Biobank were analyzed using a recently developed computational platform to identify individuals carrying KCNJ1 variants with clinical features consistent with Bartter syndrome type II. In parallel, we examined genomic data in both the NIH TOPMed and ClinVar databases with the aid of Rhapsody, a verified computational algorithm that predicts mutation pathogenicity and disease severity. Subsequent phenotypic studies using a yeast screen to assess ROMK function-and analyses of ROMK biogenesis in yeast and human cells-identified four previously uncharacterized mutations. Among these, one mutation uncovered from the two parallel approaches (G228E) destabilized ROMK and targeted it for ERAD, resulting in reduced cell surface expression. Another mutation (T300R) was ERAD-resistant, but defects in channel activity were apparent based on two-electrode voltage clamp measurements in X. laevis oocytes. Together, our results outline a new computational and experimental pipeline that can be applied to identify disease-associated alleles linked to a range of other potassium channels, and further our understanding of the ROMK structure-function relationship that may aid future therapeutic strategies to advance precision medicine.

DOI: https://doi.org/10.1371/journal.pgen.1011051
Nat Commun. 2023 11 11. 14(1): 7319

HSP47 levels determine the degree of body adiposity.

Jihoon Shin, Shinichiro Toyoda, Yosuke Okuno, Reiko Hayashi, Shigeki Nishitani, Toshiharu Onodera, Haruyo Sakamoto, Shinya Ito, Sachiko Kobayashi, Hirofumi Nagao, Shunbun Kita, Michio Otsuki, Atsunori Fukuhara, Kazuhiro Nagata, Iichiro Shimomura.

Adiposity varies among individuals with the influence of diverse physiological, pathological, environmental, hormonal, and genetic factors, but a unified molecular basis remains elusive. Here, we identify HSP47, a collagen-specific chaperone, as a key determinant of body adiposity. HSP47 expression is abundant in adipose tissue; increased with feeding, overeating, and obesity; decreased with fasting, exercise, calorie restriction, bariatric surgery, and cachexia; and correlated with fat mass, BMI, waist, and hip circumferences. Insulin and glucocorticoids, respectively, up- and down-regulate HSP47 expression. In humans, the increase of HSP47 gene expression by its intron or synonymous variants is associated with higher body adiposity traits. In mice, the adipose-specific knockout or pharmacological inhibition of HSP47 leads to lower body adiposity compared to the control. Mechanistically, HSP47 promotes collagen dynamics in the folding, secretion, and interaction with integrin, which activates FAK signaling and preserves PPARγ protein from proteasomal degradation, partly related to MDM2. The study highlights the significance of HSP47 in determining the amount of body fat individually and under various circumstances.

DOI: https://doi.org/10.1038/s41467-023-43080-x