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Abstract Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.

Abstract The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

The response of the haloarchaeal model organism Haloferax volcanii to iron starvation was analyzed at the proteome level by data-independent acquisition mass spectrometry. Cells grown in minimal medium with normal iron levels were compared to those grown under low iron conditions, with samples being separated into membrane and cytoplasmic fractions in order to focus on import/export processes which are frequently associated with metal homeostasis. Iron starvation not only caused a severe retardation of growth but also altered the levels of many proteins. Using a comprehensive annotated spectral library and data-independent acquisition mass spectrometry (DIA-MS), we found that iron starvation resulted in significant changes to both the membrane and the soluble proteomes of Hfx. volcanii. The most affected protein is the RND family permease HVO_A0467, which is 44-fold enriched in cells grown under iron starvation. The gene HVO_A0467 can be deleted suggesting that it is not essential under standard conditions. Compared to wild type cells the deletion strain shows only slight changes in growth and cell morphologies show no differences. Molecular docking predictions indicated that HVO_A0467 may be an exporter of the siderophore schizokinen for which a potential biosynthesis cluster is encoded in the Hfx. volcanii genome. Together, these findings confirm the importance of iron for archaeal cells and suggest HVO_0467 as a siderophore exporter.

IntroductionCo-fractionation mass spectrometry couples native-like separations of protein-protein complexes with mass spectrometric proteome analysis for global characterization of protein networks. The technique allows for both de novo detection of complexes and for the detection of subtle changes in their protein composition. The typical requirement for fine-grained fractionation of >80 fractions, however, translates into significant demands on sample quantity and mass spectrometric instrument time, and represents a significant barrier to experimental replication and the use of scarce sample material (ex. patient biopsies).MethodsWe developed mini-Complexome Profiling (mCP), a streamlined workflow with reduced requirements for fractionation and, thus, biological material and laboratory and instrument time. Soluble and membrane-associated protein complexes are extracted from biological material under mild conditions, and fractionated by Blue Native electrophoresis using commercial equipment. Each fraction is analysed by data-independent acquisition mass spectrometry, and known protein complexes are detected based on the coelution of known components using a novel R package with a controlled false discovery rate approach. The tool is available to the community on a GitHub repository.ResultsmCP was benchmarked using HEK293 cell lysate and exhibited performance similar to established workflows, but from a significantly reduced number of fractions. We then challenged mCP by performing comparative complexome analysis of cardiomyocytes isolated from different chambers from a single mouse heart, where we identified subtle chamber-specific changes in mitochondrial OxPhos complexes.DiscussionThe reduced sample and instrument time requirements open up new applications of co-fractionation mass spectrometry, specifically for the analysis of sparse samples such as human patient biopsies. The ability to identify subtle changes between similar tissue types (left/right ventricular and atrial cardiomyocytes) serves as a proof of principle for comparative analysis of mild/asymptomatic disease states.

Summary: How CRISPR-Cas systems defend bacteria and archaea against invading genetic elements is well understood, but less is known about their regulation. In the cyanobacterium Synechocystis sp. PCC 6803, the expression of one of the three different CRISPR-Cas systems responds to changes in environmental conditions. The cas operon promoter of this system is controlled by the light- and redox-responsive transcription factor RpaB binding to an HLR1 motif, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, the leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled with mass spectrometry analysis and site-directed mutagenesis revealed six residues involved in the CrhR-RNA interaction, with C371 being critically important. Thus, the expression of a type III-Dv CRISPR-Cas system is linked to the redox status of the photosynthetic cell at the transcriptional and post-transcriptional levels.

Abstract The regulation of thymocyte development by RNA-binding proteins (RBPs) is largely unexplored. We identify 642 RBPs in the thymus and focus on Arpp21, which shows selective and dynamic expression in early thymocytes. Arpp21 is downregulated in response to T cell receptor (TCR) and Ca2+ signals. Downregulation requires Stim1/Stim2 and CaMK4 expression and involves Arpp21 protein phosphorylation, polyubiquitination and proteasomal degradation. Arpp21 directly binds RNA through its R3H domain, with a preference for uridine-rich motifs, promoting the expression of target mRNAs. Analysis of the Arpp21–bound transcriptome reveals strong interactions with the Rag1 3′-UTR. Arpp21–deficient thymocytes show reduced Rag1 expression, delayed TCR rearrangement and a less diverse TCR repertoire. This phenotype is recapitulated in Rag1 3′-UTR mutant mice harboring a deletion of the Arpp21 response region. These findings show how thymocyte-specific Arpp21 promotes Rag1 expression to enable TCR repertoire diversity until signals from the TCR terminate Arpp21 and Rag1 activities.

Abstract If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow—and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

Summary: Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.

Abstract The reaction of CO2 with H2O to form bicarbonate (HCO3 −) and H+ controls sperm motility and fertilization via HCO3 −-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3 − in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3 − transport are not detected, and may be present at copy numbers

Abstract Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease‐specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid‐driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell‐derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V. Treatment of mutant cells with AMPK activator reestablishes fatty acid‐driven OXPHOS and protects mice against cardiac dysfunction.

Summary: RNA helicases constitute a large protein family implicated in cellular RNA homeostasis and disease development. Here, we show that the RNA helicase IGHMBP2, linked to the neuromuscular disorder spinal muscular atrophy with respiratory distress type 1 (SMARD1), associates with polysomes and impacts translation of mRNAs containing short, GC-rich, and structured 5′ UTRs. The absence of IGHMBP2 causes ribosome stalling at the start codon of target mRNAs, leading to reduced translation efficiency. The main mRNA targets of IGHMBP2-mediated regulation encode for components of the THO complex (THOC), linking IGHMBP2 to mRNA production and nuclear export. Accordingly, failure of IGHMBP2 regulation of THOC causes perturbations of the transcriptome and its encoded proteome, and ablation of THOC subunits phenocopies these changes. Thus, IGHMBP2 is an upstream regulator of THOC. Of note, IGHMBP2-dependent regulation of THOC is also observed in astrocytes derived from patients with SMARD1 disease, suggesting that deregulated mRNA metabolism contributes to SMARD1 etiology and may enable alternative therapeutic avenues.

ASCC3 is a multi-functional helicase that contains two consecutive Ski2-like helicase units. Here, the authors show that ASCC3 can unwind DNA by threading one strand of a substrate duplex through both helicase units, supported by the TRIP4 protein.

ABSTRACTFacing rapid fluctuations in their natural environment, extremophiles, like the hyperthermophilic archaeon Pyrococcus furiosus, exhibit remarkable adaptability to extreme conditions. However, our understanding of their dynamic cellular responses remains limited. This study integrates RNA-sequencing and mass spectrometry data, thereby elucidating transcriptomic and proteomic responses to heat and cold shock stress in P. furiosus. Our results reveal rapid and dynamic changes in gene and protein expression following these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, targeting a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but sustained response. Intriguingly, cold shock at 4°C elicits distinct short- and long-term responses at both RNA and protein levels. Cluster analysis identified gene sets with either congruent or contrasting trends in RNA and protein changes, representing well-separated arCOG groups tailored to their individual cellular responses. Particularly, upregulation of ribosomal proteins and significant enrichment of 5′-leadered sequences in cold-shock responsive genes suggest that translation regulation is important during cold shock adaption. Further investigating transcriptomic features, we reveal that thermal stress genes are equipped with basal sequence elements, such as strong promoter and poly(U)-terminators, facilitating a regulated response of the respective transcription units. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and providing valuable insights into the molecular adaptations that facilitate life in extreme environments.IMPORTANCEExtreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus, a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea.

Abstract Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development.

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide. Fibroblast growth factor receptor 1 (FGFR1) gene amplification is one of the most prominent and potentially targetable genetic alterations in squamous-cell lung cancer (SQCLC). Highly selective tyrosine kinase inhibitors have been developed to target FGFR1; however, resistance mechanisms originally existing in patients or acquired during treatment have so far led to limited treatment efficiency in clinical trials. In this study we performed a wide-scale phosphoproteomic mass-spectrometry analysis to explore signaling pathways that lead to resistance toward FGFR1 inhibition in lung cancer cells that display (i) intrinsic, (ii) pharmacologically induced and (iii) mutationally induced resistance. Additionally, we correlated AKT activation to CD44 expression in 175 lung cancer patient samples. We identified a CD44/PAK1/AKT signaling axis as a commonly occurring resistance mechanism to FGFR1 inhibition in lung cancer. Co-inhibition of AKT/FGFR1, CD44/FGFR1 or PAK1/FGFR1 sensitized ‘intrinsically resistant’ and ‘induced-resistant’ lung-cancer cells synergetically to FGFR1 inhibition. Furthermore, strong CD44 expression was significantly correlated with AKT activation in SQCLC patients. Collectively, our phosphoproteomic analysis of lung-cancer cells resistant to FGFR1 inhibitor provides a large data library of resistance-associated phosphorylation patterns and leads to the proposal of a common resistance pathway comprising CD44, PAK1 and AKT activation. Examination of CD44/PAK1/AKT activation could help to predict response to FGFR1 inhibition. Moreover, combination between AKT and FGFR1 inhibitors may pave the way for an effective therapy of patients with treatment-resistant FGFR1-dependent lung cancer.

Abstract Calpains are calcium-activated neutral proteases involved in the regulation of key signaling pathways. Junctophilin-2 (JP2) is a Calpain-specific proteolytic target and essential structural protein inside Ca2+ release units required for excitation-contraction coupling in cardiomyocytes. While downregulation of JP2 by Calpain cleavage in heart failure has been reported, the precise molecular identity of the Calpain cleavage sites and the (patho-)physiological roles of the JP2 proteolytic products remain controversial. We systematically analyzed the JP2 cleavage fragments as function of Calpain-1 versus Calpain-2 proteolytic activities, revealing that both Calpain isoforms preferentially cleave mouse JP2 at R565, but subsequently at three additional secondary Calpain cleavage sites. Moreover, we identified the Calpain-specific primary cleavage products for the first time in human iPSC-derived cardiomyocytes. Knockout of RyR2 in hiPSC-cardiomyocytes destabilized JP2 resulting in an increase of the Calpain-specific cleavage fragments. The primary N-terminal cleavage product NT1 accumulated in the nucleus of mouse and human cardiomyocytes in a Ca2+-dependent manner, closely associated with euchromatic chromosomal regions, where NT1 is proposed to function as a cardio-protective transcriptional regulator in heart failure. Taken together, our data suggest that stabilizing NT1 by preventing secondary cleavage events by Calpain and other proteases could be an important therapeutic target for future studies.

Alzheimer’s disease is characterized by the accumulation of aggregated tau protein. Here the authors find that Hsp chaperones, which normally protect cell homeostasis, can assemble with co-chaperones in a “multichaperone machinery” to target tau aggregation.

Cytotoxic T cells have specialised granules that are important for mediating their killing function. Here the authors characterise two types of cytotoxic granules and indicate different functions and temporal release of mediators at the immunological synapse.

Abstract The differentiation between a pulmonary metastasis and a newly developed squamous cell carcinoma of the lung in patients with prior head and neck squamous cell carcinoma (HNSCC) is difficult due to a lack of biomarkers but is crucially important for the prognosis and therapy of the affected patient. By using high‐resolution mass spectrometry in combination with stable isotope labelling by amino acids in cell culture, we identified 379 proteins that are differentially expressed in squamous cell carcinomas of the lung and the head and neck. Of those, CAV1, CAV2, LGALS1, LGALS7, CK19, and UGDH were tested by immunohistochemistry on 194 tissue samples (98 lung and 96 HNSCCs). The combination of CAV1 and LGALS7 was able to distinguish the origin of the squamous cell carcinoma with high accuracy (area under the curve 0.876). This biomarker panel was tested on a cohort of 12 clinically classified lung tumours of unknown origin after HNSCC. Nine of those tumours were immunohistochemically classifiable.

Abstract Background Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, tRNA CAG Ser $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ . Previously reported experiments have suggested that 3–5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the tRNA CAG Ser $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ . The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity. Results In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans’ strain pathogenicity or growth form. Conclusions Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the tRNA CAG Ser $$ {\mathrm{tRNA}}_{\mathrm{CAG}}^{\mathrm{Ser}} $$ might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity.

Abstract Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell-derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495–2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium.

Early steps of large 60S ribosomal subunit biogenesis are not well understood. Here, the authors combine biochemical experiments with protein-RNA crosslinking and mass spectrometry to show that the RNA helicase Dbp7 is key player during early 60S ribosomal assembly. Dbp7 regulates a series of events driving compaction of domain V/VI in early pre60S ribosomal particles.

Abstract The choroid plexus secrets cerebrospinal fluid (CSF) composed of electrolytes, cytokines, growth factors, metabolites and extracellular vesicles (EVs) that flow through the interconnected brain ventricles. On their course, CSF components can act as signals that affect, for example, neural stem cells (NSCs) residing in niches of the ventricular wall. We studied EV‐born CSF signals in an in vitro culture system. We purified EVs from the secretome of a choroid plexus cell line (Z310 cells), and from primary choroid plexus cultures and co‐cultured those EVs with NSCs isolated from the niche of the lateral and the third ventricle. EVsZ310 and EVsCHP were purified by differential centrifugation. This yielded fractions of EVs of 50–150‐nm diameter that induced a complex multicellular network formation and NSC differentiation. Both types of EV converted the round NSCs to cells that extended long processes that contacted nearby, alike‐shaped cells. Mass spectrometry showed that the differentiation‐inducing EVZ310 were enriched for membrane and membrane‐associated proteins involved in cell differentiation, membrane trafficking, and membrane organization. We hypothesize that this type of EV Z310 cargo causes changes of stem cell morphology that leads to multicellular networks in the niches. This cell‐shape transition may represent an initial step in NSC differentiation.

The TOM and TIM23 complexes facilitate the transport of nuclear-encoded proteins into the mitochondrial matrix. Here, the authors use a stalled client protein to purify the translocation supercomplex and gain insight into the TOM-TIM23 interface and the mechanism of protein handover from the TOM to the TIM23 complex.

Maturation of the ribosomal peptidyl transferase center (PTC) is mediated by universally conserved GTPases. Here, cryo-EM structures of mitochondrial ribosomal large subunit assembly intermediates and of mature ribosomes offer insight into the roles of several assembly factors, including GTPBP6’s role in both ribosome biogenesis and recycling.

Aminoglycoside antibiotics target the ribosome and induce misreading, yet which translation errors induce bacterial cell death is unclear. Here authors use quantitative mass spectrometry and show that bactericidal aminoglycosides induce clusters of errors in full-length proteins in vivo with as many as four amino acid substitutions in a row.

CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has been proposed previously. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to act upon DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to reduced survival rates following stress induction. In addition, our results indicate that Cas1 is directly involved in DNA repair as the enzymatically active site of the protein is crucial for growth under oxidative conditions. Based on biochemical assays, we propose a mechanism by which Cas1 plays a similar function to DNA repair protein Fen1 by cleaving branched intermediate structures. The present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display equivalent roles during archaeal DNA damage repair.

Cross-linking mass spectrometry (XLMS) allows mapping of protein-protein and protein-RNA interactions, but the analysis of protein-DNA complexes remains challenging. Here, the authors develop a UV light-based XLMS workflow to determine protein-DNA interfaces in reconstituted chromatin and isolated nuclei.

Comprehensive quantitative profiling of intact glycopeptides remains technically challenging. To address this, the authors here develop an integrated quantitative glycoproteomic workflow, including optimized sample preparation, multiplexed quantification and a dedicated data processing tool.

While archaeal proteomics advanced rapidly, a comprehensive proteome database for archaea is lacking. Therefore, the authors here launch the Archaeal Proteome Project, a community-effort providing insights into archaeal cell biology via the combined reanalysis of Haloferax volcanii proteomics data.

Abstract Background Dystroglycanopathies are a group of inherited disorders characterized by vast clinical and genetic heterogeneity and caused by abnormal functioning of the ECM receptor dystroglycan (Dg). Remarkably, among many cases of diagnosed dystroglycanopathies, only a small fraction can be linked directly to mutations in Dg or its regulatory enzymes, implying the involvement of other, not-yet-characterized, Dg-regulating factors. To advance disease diagnostics and develop new treatment strategies, new approaches to find dystroglycanopathy-related factors should be considered. The Dg complex is highly evolutionarily conserved; therefore, model genetic organisms provide excellent systems to address this challenge. In particular, Drosophila is amenable to experiments not feasible in any other system, allowing original insights about the functional interactors of the Dg complex. Methods To identify new players contributing to dystroglycanopathies, we used Drosophila as a genetic muscular dystrophy model. Using mass spectrometry, we searched for muscle-specific Dg interactors. Next, in silico analyses allowed us to determine their association with diseases and pathological conditions in humans. Using immunohistochemical, biochemical, and genetic interaction approaches followed by the detailed analysis of the muscle tissue architecture, we verified Dg interaction with some of the discovered factors. Analyses of mouse muscles and myocytes were used to test if interactions are conserved in vertebrates. Results The muscle-specific Dg complexome revealed novel components that influence the efficiency of Dg function in the muscles. We identified the closest human homologs for Dg-interacting partners, determined their significant enrichment in disease-associations, and verified some of the newly identified Dg interactions. We found that Dg associates with two components of the mechanosignaling Hippo pathway: the WW domain-containing proteins Kibra and Yorkie. Importantly, this conserved interaction manages adult muscle size and integrity. Conclusions The results presented in this study provide a new list of muscle-specific Dg interactors, further analysis of which could aid not only in the diagnosis of muscular dystrophies, but also in the development of new therapeutics. To regulate muscle fitness during aging and disease, Dg associates with Kibra and Yorkie and acts as a transmembrane Hippo signaling receptor that transmits extracellular information to intracellular signaling cascades, regulating muscle gene expression.

Cyclin-dependent kinases (CDKs) are key players in cell cycle regulation and transcription. The CDK-family member Cdk10 is important for neural development and can act as a tumour suppressor, but the underlying molecular mechanisms are largely unknown. Here, we provide an in-depth analysis of Cdk10 substrate specificity and function. Using recombinant Cdk10/CycQ protein complexes, we characterize RNA pol II CTD, c-MYC and RB1 as in vitro protein substrates. Using an analogue-sensitive mutant kinase, we identify 89 different Cdk10 phosphosites in HEK cells originating from 66 different proteins. Among these, proteins involved in cell cycle, translation, stress response, growth signalling, as well as rRNA, and mRNA transcriptional regulation, are found. Of a set of pan-selective CDK- and Cdk9-specific inhibitors tested, all inhibited Cdk10/CycQ at least five times weaker than their proposed target kinases. We also identify Cdk10 as an in vitro substrate of Cdk1 and Cdk5 at multiple sites, allowing for a potential cross-talk between these CDKs. With this functional characterization, Cdk10 adopts a hybrid position in both cell cycle and transcriptional regulation.

Abstract Introduction Mutations in pre‐mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri‐snRNP complex, cause autosomal‐dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri‐snRNP proteins result in retina‐specific disorders, and so far, the underlying mechanism of splicing factors‐related RP is poorly understood. Methods We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31‐adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. Results To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle‐related pathways to be significantly affected. Strikingly, the patient‐derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin‐conjugated proteins including key visual cycle and other RP‐linked tri‐snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild‐type levels led to tri‐snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. Conclusions Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31‐initiated mis‐splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31‐adRP patients.

The Staufen proteins recognize secondary structures in 3’-untranslated regions in mRNA transcripts and induce degradation of these mRNAs with the help of the RNA helicase UPF1. Here the authors report that the nonsense-mediated mRNA decay factor UPF2 mediates the interaction between Stau1 and UPF1 in Staufen-mediated mRNA decay.

The NER machinery contains the multisubunit transcription factor IIH (TFIIH) that opens the DNA repair bubble, scans for the lesion, and coordinates excision of the damaged site. Here the authors resolve the cryo-electron microscopy structure of the human core TFIIH-XPA-DNA complex and provide insights into its activation.

α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson’s disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked hαSynTP or hαSynΔ119 (h stands for “human”) into the murine αSyn locus. hαSynTP is a structure-based mutant with triple alanine to proline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of hαSynTP or hαSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that hαSynTP and hαSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSynTP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSynTP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.

To assemble a brain, differentiating neurons must make proper connections and establish specialized brain compartments. Abnormal levels of cell adhesion molecules disrupt these processes. Dystroglycan (Dg) is a major non-integrin cell adhesion receptor, deregulation of which is associated with dramatic neuroanatomical defects such as lissencephaly type II or cobblestone brain. The previously established Drosophila model for cobblestone lissencephaly was used to understand how Dg is regulated in the brain. During development, Dg has a spatiotemporally dynamic expression pattern, fine-tuning of which is crucial for accurate brain assembly. In addition, mass spectrometry analyses identified numerous components associated with Dg in neurons, including several proteins of the exocyst complex. Data show that exocyst-based membrane trafficking of Dg allows its distinct expression pattern, essential for proper brain morphogenesis. Further studies of the Dg neuronal interactome will allow identification of new factors involved in the development of dystroglycanopathies and advance disease diagnostics in humans.

Pre-ribosomes undergo numerous structural rearrangements during their assembly. Here the authors identify the binding sites of three essential RNA helicases on pre-ribosomal particles, enabling them to provide insights into the structural and compositional changes that occur during biogenesis of the large ribosomal subunit.

Mutations in pre-mRNA processing factors cause autosomal dominant retinitis pigmentosa. Here the authors provide insights into the pathophysiological mechanisms underlying non-syndromic retinal disease caused by heterozygous mutations in genes encoding ubiquitously expressed splicing factors.

Measuring precise protein turnover rates in animals is technically challenging at the proteomic level. Here, Fornasiero and colleagues use isotopic labeling with mass spectrometry and mathematical modeling to accurately determine protein lifetimes in the mouse brain

The chaperone Hsp90 plays a key role in maintaining cellular homeostasis. Here the authors provide structural insights into substrate recognition and the pro-folding mechanism of Hsp90/co-chaperone complexes by studying the complex of Hsp90 with its co-chaperone FKBP51 and the substrate Tau bound Hsp90/FKBP51 ternary complex using a NMR based integrative approach.

Abstract Patients with head‐and‐neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head‐and‐neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross‐ and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC.

The Paf1 complex (Paf1C) is an elongation factor assembly that forms the interface between transcribing Pol II and chromatin factors. Here the authors describe the architecture of Paf1C and its interface with Pol II, and show that Paf1C is globally required for normal mRNA transcription in yeast.

Targeting CD8+ T cells to recurrent tumor-specific mutations can profoundly contribute to cancer treatment. Some of these mutations are potential tumor antigens although they can be displayed by non-spliced epitopes only in a few patients, because of the low affinity of the mutated non-spliced peptides for the predominant HLA class I alleles. Here, we describe a pipeline that uses the large sequence variety of proteasome-generated spliced peptides and identifies spliced epitope candidates, which carry the mutations and bind the predominant HLA-I alleles with high affinity. They could be used in adoptive T cell therapy and other anti-cancer immunotherapies for large cohorts of cancer patients. As a proof of principle, the application of this pipeline led to the identification of a KRAS G12V mutation-carrying spliced epitope candidate, which is produced by proteasomes, transported by TAPs and efficiently presented by the most prevalent HLA class I molecules, HLA-A*02:01 complexes.

Alzheimer’s disease is the most prevalent neurodegenerative disorder leading to progressive cognitive decline. Despite decades of research, understanding AD progression at the molecular level, especially at its early stages, remains elusive. Here, we identified several presymptomatic AD markers by investigating brain proteome changes over the course of neurodegeneration in a transgenic mouse model of AD (3×Tg-AD). We show that one of these markers, heme-binding protein 1 (Hebp1), is elevated in the brains of both 3×Tg-AD mice and patients affected by rapidly-progressing forms of AD. Hebp1, predominantly expressed in neurons, interacts with the mitochondrial contact site complex (MICOS) and exhibits a perimitochondrial localization. Strikingly, wildtype, but not Hebp1-deficient, neurons showed elevated cytotoxicity in response to heme-induced apoptosis. Increased survivability in Hebp1-deficient neurons is conferred by blocking the activation of the mitochondrial-associated caspase signaling pathway. Taken together, our data highlight a role of Hebp1 in progressive neuronal loss during AD progression.

The assembly of the splicesome involves several distinct stages that require the sequential action of DExD/H-box RNA helicases. Here, the authors uncover a new intermediate, the pre-B complex, that accumulates in the presence of an inactive form of the DEAD-box protein Prp28.

Heterochromatin protein 1 (HP1), including HP1 α, β and γ, is a family of non-histone chromatin factors thought to be involved in chromatin organization. Here, the authors show that dimeric HP1β interacts dynamically with H3K9me3, a hallmark of heterochromatin, and bridges condensed chromatin.