Biology subjects

Varjosalo, M.

Publications and source records attributed to Varjosalo, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Conjunctive Targeting Links Drug Synergy to Emergent Proteome Structural States

Combinatorial therapies are widely used in the treatment of acute myeloid leukemia (AML) to address disease heterogeneity, adaptive resistance, and rewired signaling and metabolic states. Yet drug prioritization remains largely guided by clinical or phenotypic evidence, while the molecular mechanisms underlying effective drug combinations remain incompletely defined. To narrow this gap, we developed Combinatorial high-ratio Partial proteolysis with reference PRoteome Analysis (CoPPRA), a structural proteomics workflow based on limited proteolysis of cell lysates that profiles drug-associated changes in regional protein accessibility at peptide-level resolution. Here, we applied CoPPRA to ruxolitinib and ulixertinib, individually and in combination, in AML-related cell lysates. Our findings extend conjunctive targeting (CT), a recently proposed mechanism of combinatorial drug action in which combined exposure produces protein targeting patterns not observed with either drug alone. Previously identified through combination-associated changes in protein solubility/stability, CT is examined here at peptide-level resolution through regional differences in proteolytic accessibility. The ruxolitinib-ulixertinib combination produced broad peptide-level accessibility changes, including a subset meeting the predefined criteria for CT. CT candidates predominantly exhibited regional accessibility changes, with altered peptide regions occurring against comparatively small changes across the remaining quantified peptides from the same proteins. MAP2K1 and ATP6V1G1 showed pronounced differences between overlapping peptide sequences, highlighting localized variation in combination-associated accessibility, including an ATP6V1G1 peptide mapping to an annotated helical region. Combination-associated increases in peptide signals were also observed in PIK3R1, BRD4, and PTPN11, linking regional accessibility changes to signaling and transcriptional regulators relevant to AML. Functional enrichment and network analyses further implicated nucleotide and glucose metabolism, ficolin-1-rich granules, ribosome-associated processes, and phagocytic vesicles. These results extend conjunctive targeting from protein-level solubility/stability changes to regional differences in proteolytic accessibility, showing that combination-associated effects can be concentrated within specific peptide regions rather than distributed uniformly across proteins. More broadly, CoPPRA provides a peptide-resolved approach for investigating the molecular features of combinatorial drug action and prioritizing protein regions for subsequent mechanistic validation.

biochemistry↗

bioRxiv2026

Chemical Proteomics of Residual Acute Myeloid Leukemia Cells Reveals Therapeutic Vulnerabilities

Venetoclax combined with azacitidine has improved treatment outcomes in acute myeloid leukemia (AML), yet relapse and treatment persistence remain major clinical challenges. To investigate proteomic mechanisms associated with venetoclax-azacitidine response and adaptation, we applied a multi-layer combinatorial proteome integral solubility/stability alteration analysis (CoPISA) strategy in SKM-1 AML cells. Cells were treated with venetoclax, azacitidine, their combination, or vehicle control and profiled across four orthogonal layers: short-term lysate CoPISA, short-term intact-cell CoPISA, long-term intact-cell CoPISA after 5 days of treatment, and long-term expression proteomics of surviving cells. The venetoclax-azacitidine combination induced treatment-specific protein solubility and abundance changes that were not fully reproduced by either single agent. Long-term surviving cells displayed extensive proteomic remodeling, consistent with the emergence of an adaptive drug-tolerant state, although contributions from pre-existing resilient cell populations cannot be excluded. Integration of short- and long-term solubility changes with abundance remodeling revealed distinct adaptive regimes, including retained biochemical targets, dosage-compensated targets, sensitive-state-specific targets, and remodeled adaptive targets. This framework prioritized candidate resistance-associated proteins, including NRP2, RPL18, PLP2, RPS28, and NOTCH1. NRP2 emerged as a top candidate across all proteomics layers, consistently showing increased abundance. Overall, this study shows that combining CoPISA with expression proteomics can resolve temporally distinct proteomic states of venetoclax-azacitidine response and identify candidate adaptive vulnerabilities in AML.

cancer biology↗

bioRxiv2026

A novel method for obtaining epileptic brain tissue for omic analyses using electrodes from a clinical stereoelectroencephalography study

Abstract Purpose: Developing a valid and practical method for obtaining brain tissue samples for transcriptomic and proteomic analyses using tissue adhered to stereoelectroencephalography (SEEG) electrodes, to enable investigation of the molecular mechanisms underlying chronic epilepsy. Method: Brain tissue samples adhered to SEEG electrodes were collected from six patients with drug-resistant epilepsy and preprocessed in a hospital environment after electrode removal. RNA extraction was initiated immediately, and proteomics samples were snap-frozen until subsequent analysis. The samples were categorized into three groups according to their electrophysiological profile: epileptogenic zone, propagation zone, and least-involved zone. The omic findings between these zones and anatomical brain areas were compared. Results: High-quality RNA and protein samples were obtained from tissue adhered to SEEG electrodes. Neuron- and brain-specific gene expression patterns and proteins were identified. Signs of activation of inflammatory mechanisms were most pronounced in the epileptogenic zone. Transcriptomic and proteomic findings demonstrated concordance. Conclusion: SEEG electrodes are a useful source for obtaining brain tissue for molecular characterization of chronic epilepsy. This method will enable the identification of shared and distinct molecular mechanisms in patients with varying etiologies of epilepsy.

neuroscience↗

bioRxiv2026

β4-integrins safeguard nuclear mechanics to suppress prostate cancer progression

Prostate cancer (PCa) progression is accompanied by profound alterations in cell-extracellular matrix (ECM) adhesion, nuclear architecture and mechanical adaptability, yet the molecular mechanisms linking these processes remain poorly understood. Hemidesmosomes (HDs), formed by 6{beta}4-integrins, anchor epithelial cells to the basement membrane and couple extracellular forces to the intermediate filament (IF) cytoskeleton. Here, we identify a previously unrecognized tumor-suppressive function of {beta}4-integrins in preserving nuclear integrity in prostate epithelial cells. Loss of {beta}4-integrins disrupted the cytokeratin-5 network and its coupling to the nucleus, leading to nuclear softening, lamin remodeling, reduced heterochromatin content and enhanced confined migration. Unexpectedly, proximity-labeling proteomics revealed that {beta}4-integrins engage nuclear pore complex (NPC) components in an 6-independent manner, particularly upon HD disassembly. Selected interactions were validated using proximity ligation and co-immunoprecipitation assays. {beta}4-integrin loss was associated with enlarged nuclear pores and aberrant nucleocytoplasmic transport, including nuclear accumulation of YAP1. Consistent with these findings, reduced {beta}4-integrin expression in a large PCa tissue cohort correlated with altered nuclear morphology, adverse clinicopathological features, metastatic progression, and poor patient survival. Collectively, our study establishes {beta}4-integrins as a critical molecular link between cell-ECM adhesion, nuclear mechanics and genome integrity.

cancer biology↗

bioRxiv2026

Multi-layered regulatory networks driving human dopaminergic neuron differentiation

Neuronal differentiation requires coordinated regulation across chromatin organization, gene expression, protein abundance, and post-translational modifications. Using the LUHMES human dopaminergic neuronal differentiation model, we integrated proteome and phosphoproteome profiling with previously generated enhancer-promoter interaction maps from NET-CAGE and HiCap and transcriptomic analysis across three consecutive differentiation stages. Differentiation was accompanied by increased abundance and phosphorylation of proteins involved in axon guidance, cytoskeletal organization, and synaptic signaling, alongside repression of the cell cycle, DNA replication, and chromatin-associated programs. Phosphoproteome analysis further revealed extensive remodeling of signaling networks associated with neuronal maturation. Enhancer-promoter interaction analysis revealed substantially greater rewiring at enhancers than promoters and identified master and relay transcription factors regulated across multiple molecular layers. siRNA-mediated knockdown showed that transcription factors MYT1, ISL2, and NHLH2 are crucial for proper neuronal maturation, whereas LCOR acts as a negative regulator of differentiation. Integrative network reconstruction further nominated MEOX2 as a candidate enhancer-associated regulator of late dopaminergic maturation. Together, these findings provide a multi-layered view of regulatory networks governing human neuronal differentiation.

systems biology↗

bioRxiv2026

A phosphorylation switch in PAGE4 drives MED12-mutant fibroid pathogenesis

Recurrent somatic mutations in MED12, found in [~]70% of uterine leiomyomas (ULs), define the dominant molecular subtype of these highly prevalent tumors, yet the downstream effector mechanisms remain poorly understood. Using an integrated multi-omics workflow, encompassing discovery-phase DDA proteomics of matched leiomyoma-myometrium pairs, validation-phase DIA proteomics across genetically stratified cohorts (MED12 p.G44D, RAD51B-HMGA2, IRS4/FH subtypes), phosphoproteomics, immunohistochemistry, and AP-MS interactomics, we identify prostate-associated gene 4 (PAGE4) as a central effector of MED12-mutant UL pathogenesis. PAGE4 emerged as one of the most significantly upregulated proteins in MED12-mutant ULs and harbored the highest-occupancy hyperphosphorylation sites (T51, T85) in the tumor phosphoproteome, a pattern confirmed by immunohistochemistry and orthogonal phosphoproteomics. Kinase-substrate enrichment nominated HIPK2 as the primary upstream kinase, with supporting evidence from the broader CMGC family. Subsequent analysis revealed that phosphorylation acts as a molecular switch, fundamentally restructuring the PAGE4 interactome to favor the Mediator complex and RNA Pol II transcriptional machinery. This rewiring was functionally validated by ChIP-seq and luciferase reporter assays, which demonstrated corresponding shifts in transcription factor occupancy and downstream pathway activity. Collectively, these data establish phospho-PAGE4 as a critical mechanistic node downstream of MED12 mutation, expand PAGE4 biology from prostate cancer to female reproductive tumors, and nominate it as a mechanistic biomarker and candidate therapeutic vulnerability in the most prevalent molecular subtype of uterine leiomyomas.

systems biology↗

bioRxiv2026

Efficient NK cell transduction with VSV-G-pseudotyped lentiviral vectors

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors such as NK cell activation, construct design, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped LVs for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with a NK cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and Retronectin, resulting in excellent transduction efficiency without compromising NK cell phenotype or growth. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

immunology↗

bioRxiv2025

ISG15 Differentially Modulates Clade Ib and II MPXV Infection in MEF cells

The unprecedented human-to-human transmission of Clade IIb monkeypox virus (MPXV) during the 2022 outbreak has renewed focus on host determinants of viral fitness. Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein with broad immunomodulatory functions, yet its role in MPXV infection remains unclear. Using representative strains from recent and historical outbreaks spanning Clades I and II, we show that ISG15 deficiency enhances viral replication and protein production in murine cells. Given that rodents are considered potential natural reservoirs of MPXV, these findings highlight the importance of studying murine models to understand virus-host interactions. Notably, the 2024 Democratic Republic of Congo strain displays reduced sensitivity to ISG15, suggesting clade-specific adaptation. ISG15 also influences viral immune evasion, as knockout cells infected with Clade II viruses expressed fewer immunomodulatory proteins and exhibited marked reductions in host protein phosphorylation. These results identify ISG15 as a determinant of MPXV infection and underscore evolutionary differences between clades.

microbiology↗

bioRxiv2025

Automated MagNet Enrichment Unlocks Deep and Cost-Effective LC-MS Plasma Proteomics

Plasma is an ideal material for proteomics due to its diverse protein content reflecting physiological and pathological states, and its compatibility with minimally invasive sampling. Deep proteomic profiling of plasma is hindered by the dominance of high-abundant proteins that mask the detection of low-abundant proteins. To overcome this, we compared five plasma protein enrichment methods, MagNet, ENRICHplus, ENRICHiST, EasySep, and EXONET, against neat plasma using LC-MS proteomics. All five methods substantially increased protein identifications, with MagNet, ENRICHplus, EasySep, and EXONET yielding up to 4200 proteins per sample, over 7-fold more than neat plasma, using a 44-minute gradient on the Evosep One and data-independent acquisition on the timsTOF Pro 2. These methods enriched extracellular vesicle-associated proteins while effectively depleting high-abundant proteins. We further optimized the cost-effective MagNet protocol by increasing the plasma-to-bead ratio and automated the workflow, including Evotip loading, on the Biomek i5 liquid handler. Automated MagNet demonstrated high reproducibility and a remarkably low total cost of just a few dollars per sample. This streamlined enrichment strategy enables scalable, high-throughput LC-MS plasma proteomics, supporting biomarker discovery across large clinical cohorts.

systems biology↗

bioRxiv2025

The non-catalytic ϵ DNA polymerase subunit is an NPF motif recognition protein

Short linear motifs (SLiMs) in disordered protein regions direct numerous protein-protein interactions, yet most remain uncharacterized. The Asn-Pro-Phe (NPF) motif is a well-known EH-domain ligand implicated in endocytosis, but here we reveal that the non-catalytic subunit of human DNA polymerase {varepsilon} (POLE2) also serves as a general NPF-motif receptor. Using a quantitative "native holdup" assay, we find that POLE2 selectively binds diverse NPF-containing peptides, including canonical EH-domain ligands (e.g., SYNJ1) and previously uncharacterized motifs. Biochemical measurements and mutational analysis show that NPF motifs interact with a shallow pocket near the POLE2 C-terminus, and AlphaFold predictions confirm key roles for Y513, E520, and S522 in motif coordination. Proteome-scale affinity screens identify NPF-containing nuclear proteins (e.g., WDHD1, DONSON, TTF2) that bind POLE2 with micromolar affinities, and their motif mutations abolish binding in cell extracts. Although POLE2 primarily tethers the catalytic POLE subunit to replication forks, these results indicate that it can also recruit various NPF-bearing partners involved in replication, DNA repair, and transcription regulation. Notably, NPF motifs optimized for EH-domain binding can still associate with POLE2, highlighting the inherent degeneracy of SLiM-mediated networks. Overall, these findings establish POLE2 as a central hub linking replication with other processes via broad NPF-motif recognition.

biochemistry↗

bioRxiv2025

The novel SCN5A-P1891A mutation is associated with left ventricular hypertrabeculation and links Nav1.5 to cardiomyocyte proliferation and disrupted 3D cardiac tissue formation

BackgroundLeft ventricular hypertrabeculation (LVHT) is a heterogenous cardiac condition with a complex and poorly understood aetiology. We comprehensively characterised the effect of a novel P1891A mutation in the SCN5A gene, which encodes the voltage-gated sodium channel Nav1.5, identified in a Finnish family diagnosed with LVHT. MethodsWe generated SCN5A-P1891A mutation-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs) and performed electrophysiological assessments, including patch-clamp studies, and fluorescent calcium imaging, to determine the mutations effect on hiPSC-CM electrophysiology. We also evaluated the impact of the mutation on the proliferative capacity in response to mitogenic stimuli and on the hypertrophic response following cyclic mechanical stretch or endothelin-1 treatment. Further, we assessed the effect on contractile parameters in three-dimensional (3D) contractile hydrogels (engineered heart tissues, EHTs) and conducted advanced proteomics to understand the consequences of the mutation on Nav1.5 protein-protein interactions. ResultsThe SCN5A-P1891A mutation reduced the sodium current densities and increased both the sodium window current and arrhythmogenicity; however, action potential parameters were unaffected. Advanced proteomics characterised, for the first time, the complete Nav1.5 interactome and revealed that the SCN5A-P1891A mutation negated interactions with fibroblast growth factor 12 (FGF12) and FGF13, that are known to modulate sodium channel activity. Baseline proliferation was unchanged, although aged P1891A-hiPSC-CMs demonstrated enhanced proliferative capacity following mitogenic stimulation. Further, P1891A-hiPSC-CMs exhibited a heightened stress response upon mechanical stretch, resulting in the upregulation of heart failure-associated genes. Strikingly, EHTs derived from P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst the majority condensed only partially and failed to beat synchronously, a small subset condensed fully yet exhibited weak contractile properties, alongside age-associated functional decline. In contrast, EHTs derived from healthy control hiPSC-CMs consistently condensed fully and demonstrated a positive correlation between post-fabrication age and contractile properties ConclusionsOur study presents a unique aetiology of LVHT and reveals a novel association between SCN5A mutations and enhanced human cardiomyocyte proliferation. Further, the inability of P1891A-hiPSC-CMs to consistently form fully condensed 3D cardiac tissues may be linked to their abnormal response to mechanical stretch and provides a powerful 3D model for future mechanistic research and drug development studies to better understand and treat LVHT.

molecular biology↗

bioRxiv2024

Human skeletal muscle possesses both reversible proteomic signatures and a retained proteomic memory after repeated resistance training

Investigating repeated resistance training separated by a training break enables exploration of the potential for a proteomic memory of resistance training (RT)-induced skeletal muscle growth. Our aim was to examine skeletal muscle proteome response to 10-week RT (RT1) followed by 10-week training cessation (i.e. detraining, DT), and finally, 10-week retraining (RT2). Thirty healthy, untrained participants conducted either periodic RT (RT1-DT-RT2, n=17) or a 10-week no-training control period (n=13) followed by 20 weeks of RT (n=11). RT included twice-weekly supervised whole-body RT sessions, and resting vastus lateralis biopsies were obtained every ten weeks for proteomics analysis using high-end DIA-PASEFs mass spectrometry. The first RT period altered 150 proteins (93% increased) involved in e.g. energy metabolism and protein processing compared with minor changes during the no-training control period. The proteome adaptations were similar after the second RT compared to baseline demonstrating reproducibility in proteome adaptations to RT. Many of the proteins induced by RT1 were reversed towards baseline after detraining and increased again after retraining. These reversible proteins were especially involved in aerobic energy metabolism. Interestingly, several proteins increased after RT1 remain elevated after detraining, including carbonyl reductase 1 (CBR1) and proteins involved in muscle contraction, cytoskeleton and calcium-binding. Amongst the latter, calcium-activated protease calpain-2 (CAPN2) has been recently identified as an epigenetic muscle memory gene. We show that resistance training evokes retained protein levels even after 2.5 months of no training. This is the first study to demonstrate a potential proteomic memory of resistance training-induced muscle growth in human skeletal muscle. Key pointsO_LIRepeated resistance training in humans separated by a training break (i.e. detraining) enables the identification of temporal protein signatures over the training, detraining, and retraining periods as well as studying reproducibility of protein changes to resistance training. C_LIO_LIMuscle proteome adaptations were similar after a second period of resistance training when compared to baseline, demonstrating reproducibility in proteome adaptations to earlier resistance training. C_LIO_LIMany of the proteins induced by resistance training were reversed towards baseline after detraining and increased again after retraining. These reversible proteins were especially involved in aerobic energy metabolism. C_LIO_LISeveral proteins increased after resistance training remain elevated after detraining, including carbonyl reductase 1 (CBR1) and calcium-binding proteins such as calpain-2 (CAPN2), a recently identified epigenetic muscle memory gene. C_LIO_LIHuman skeletal muscle experiences retained protein changes following resistance training persisting over two months demonstrating a potential proteomic memory of resistance training-induced muscle growth. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/624068v3_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1f0ae2corg.highwire.dtl.DTLVardef@3af3b4org.highwire.dtl.DTLVardef@55d064org.highwire.dtl.DTLVardef@1e46bf9_HPS_FORMAT_FIGEXP M_FIG Human skeletal muscle proteome response was investigated after 10-week resistance training (RT1) followed by 10-week training cessation (i.e. detraining, DT), and finally, 10-week retraining (RT2). Many of the proteins were reversed towards baseline after DT and increased again after RT2. These reversible proteins were especially involved in aerobic energy metabolism. However, several RT-induced proteins remain elevated after DT, including carbonyl reductase 1 (CBR1) and many proteins involved in muscle contraction or cytoskeleton and calcium-binding. Amongst the latter, calcium-activated protease calpain-2 (CAPN2) is a recently identified epigenetic muscle memory gene. This study shows that resistance training evokes retained protein levels even after 2.5 months of no training and demonstrates a potential proteomic memory of RT-induced muscle growth in human skeletal muscle. Created in BioRender.com. C_FIG

physiology↗

bioRxiv2024

T cell correction pipeline for Inborn Errors of Immunity

CRISPR/Cas9 gene editing technology is a promising tool for correcting pathogenic variants for autologous cell therapies for Inborn Errors of Immunity (IEI). The present IEI correction strategies mainly focus on the knock-in of therapeutic cDNAs, or knockout of the disease-causing gene when feasible. These strategies address many single-gene defects but may disrupt gene expression and require significant optimization for each newly discovered IEI-causing gene, highlighting the need for complementary platforms that can precisely correct diverse pathogenic variants. Here, we present a safe and efficient T cell single nucleotide variant (SNV) correction pipeline based on homology-directed repair (HDR), suitable for diverse monogenic mutations. By using founder mutations of Deficiency of ADA2 (DADA2), Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and Cartilage Hair Hypoplasia (CHH) as IEI models, we show that our pipeline can achieve up to 80% bi-allelic editing, with resultant functional correction of the disease phenotype in patient T cells. We do not find detectable pre-malignant off-target effects or karyotypic, transcriptomic or proteomic aberrations upon profiling patient T cells with GUIDE-seq, single cell RNA sequencing, PacBio based long-read whole genome sequencing, and high-throughput proteomics. This study demonstrates that HDR-based SNV editing is a safe and effective option for IEI T cell correction and that it could be developed to an autologous T cell therapy, as the presented protocol is scalable for a GMP-compatible workflow. This study is a step towards the development of gene correction platform that targets a broad number of monogenic mutations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/610811v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f1eb47org.highwire.dtl.DTLVardef@18dbcc2org.highwire.dtl.DTLVardef@63862dorg.highwire.dtl.DTLVardef@1fe1561_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG

bioengineering↗

bioRxiv2024

FINCA disease mouse model exhibits altered behaviour and immune response

Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA) is a childhood-onset multi-organ neurodevelopmental disorder associated with multi-organ manifestations and recurrent infections. The disease is caused by variants in NHLRC2 initiating a cascade of unknown pathological events. Previously, we have demonstrated that despite the significant decrease at the molecular level, the compound heterozygosity of knock out and p.Asp148Tyr alleles in NHLRC2 does not lead to a severe phenotype in mice. Here, we analysed the behavioural and immunological phenotype of the FINCA mice and studied the molecular pathways affected by p.Asp148Tyr in NHLRC2 using mouse and human-derived cell culture models. The FINCA mice displayed a mild hyperactivity and deficient early immune response when challenged with LPS leading to altered cytokine responses, including IFN{gamma}, IL-12, and TNF. By comparing gene expression and putative interaction partners affected by p.Asp148Tyr, we identified Rho GTPase signalling as the common pathway. Altogether, these results establish a multi-dimensional impact of the p.Asp148Tyr variant in NHLRC2. Knowledge of the molecular pathways affected by NHLRC2 and the natural course of FINCA disease progression are instrumental for the development of effective therapeutics. Summary statementFINCA is a paediatric neurodevelopmental and multi-organ disorder caused by variants in NHLRC2. Here, mild hyperactivity in connection with altered early immune response is described in the FINCA mouse model.

neuroscience↗

bioRxiv2023

Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells

The Kelch-like ECH-associated protein 1 (KEAP1) - Nuclear factor erythroid 2-related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNF) - Nuclear Factor kappa subunit B (NF{kappa}B) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and the mutants was employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NF{kappa}B activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NF{kappa}B pathway, thereby protecting against apoptosis.

cancer biology↗

bioRxiv2023

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

In this work, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3 splice site usage. Together, our results highlight the role 3-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.

molecular biology↗

bioRxiv2023

GTPBP8 is required for mitoribosomal biogenesis and mitochondrial translation

Mitochondria contain a multi-copy genome and a distinct set of ribosomes devoted to the exclusive synthesis of proteins that are essential for oxidative phosphorylation. The assembly of mitoribosomes for mitochondrial translation is a poorly understood process. In this study, we identify the uncharacterized GTP-binding protein 8 (GTPBP8) as a mitoribosomal assembly factor that specifically associates with the mitoribosomal large subunit. Genetic depletion of GTPBP8 causes an aberrant accumulation of the large mitoribosomal subunit at a late assembly stage and reduces the level of fully assembled 55S mitoribosomes, resulting in impaired mitochondrial translation and function. Together, our findings uncover an important role for human GTPBP8 in the processes of mitoribosomal assembly and mitochondrial translation. Key pointsO_LIGTPBP8 is a novel GTPase residing in the matrix peripherally bound to the inner mitochondrial membrane C_LIO_LIGTPBP8 specifically associates with the large mitoribosome subunit through interactions with large mitoribosomal proteins assembled at late stages. C_LIO_LIGTPBP is essential for maturation of the mitoribosomal large subunit and monosome formation C_LI

cell biology↗

bioRxiv2023

Structure and Interactions of the Endogenous Human Commander Complex

The Commander complex, a 16-protein subunit assembly, plays multiple roles in various intracellular events, including regulation of cell homeostasis, cell cycle, and immune response. The complex is composed of COMMD1-10, CCDC22, CCDC93, DENND10, VPS26C, VPS29, and VPS35L. These proteins are expressed ubiquitously in the human body and have been linked to diseases including Wilsons disease, atherosclerosis, and several cancers. Despite its importance, the structure and molecular functions of the Commander complex are poorly understood. Here, we report the structure and key interactions of the endogenous human Commander complex by cryogenic electron microscopy (cryo-EM) and mass spectrometry-based proteomics. Our results show that the complex is asymmetric, consisting of a stable core of a pseudo-symmetric ring of COMMD proteins 1-10 and a mobile effector consisting of DENND10 and the Retriever sub-complex, constituted by VPS35L, VPS29 and VPS26C. The two halves are scaffolded together by CCDC22 and CCDC93. This study directly confirms the cellular composition of Commander and identifies major interaction interfaces, defining the structure and interaction landscape of the complex. These findings offer new insights into its known roles in endosomal processes and intracellular transport, and uncovers a strong association with cilium assembly, and centrosome and centriole functions. HighlightsO_LIHigh-resolution structure of the Commander complex determined by cryo-EM shows a rigid COMMD1-10 core decorated by mobile effectors DENND10 and the Retriever sub-complex. C_LIO_LIComprehensive interactome analysis uncovers a plethora of novel interactions, implicating the Commander complex in previously unidentified processes, such as cilium assembly, cell cycle, and organelle biogenesis. C_LIO_LIStructural organization and identified interaction partners of the Commander complex provide a basis for further research into its molecular functions, related diseases, and potential therapeutic targets. C_LI eTOC blurbWe provide a comprehensive structural analysis of the endogenous human Commander complex, revealing its functional organization and novel interactions involved in diverse cellular processes. These findings uncover new interaction surfaces and pave the way for further research into the molecular mechanisms of the Commander complex.

biophysics↗