Biology subjects

Schilling, O.

Publications and source records attributed to Schilling, O..

At least 19 recordsLinked to original sources

bioRxiv2026

Distinct effects of hypomorphic IFT and dynein-2 skeletal ciliopathy disease alleles on chondrogenic differentiation, ECM composition and wnt signalling in ATDC5 derived cartilage-like organoids

Dysfunction of ciliary intraflagellar transport (IFT) and dynein-2 genes causes severe developmental defects, including skeletal phenotypes characterized by shortened ribs and long bones and polydactyly. Specific gene-phenotype associations suggest individual functions of the different IFT/dynein-2 proteins in development. Since null models disrupt ciliogenesis and hence are not suitable to study individual protein functions, we recreated human hypomorphic disease alleles in IFT-A (IFT43 p.M1V), IFT-B (IFT74 deletion of exon 2), and dynein-2 (WDR60 p.A911V), alongside a WDR60 null model in ATDC5 chondrocyte precursor cells. Hypomorphic mutants did not show alterations in ciliation efficiency or cilia length but displayed distinct defects in IFT88 localization, indicating impaired intraflagellar transport. Despite altered IFT, Hedgehog signalling responses were variably affected across the different genotypes. Transcriptomic analysis revealed concurrent increases in canonical Wnt signalling and expression of genes related to late skeletal development in WDR60 A911V and IFT74 del ex 2 mutants, but not in IFT43 M1V or WDR60 loss-of-function mutants. These changes were accompanied by alterations in ECM composition. In addition, all hypomorphic mutants showed reduced levels of the non-canonical Wnt ligand WNT5A in ECM proteomic analyses. Interestingly, loss of cilia in WDR60 loss-of-function mutants had only modest effects on chondrogenic differentiation and ECM composition. Overall, our data provide new evidence of genotype-dependent altered ECM composition as well as dysregulation of canonical and non-canonical Wnt signalling and accelerated chondrocyte differentiation in skeletal ciliopathies. Furthermore, our findings suggest a modulatory rather than essential role of the primary cilium for ATDC5 cell differentiation.

cell biology↗

bioRxiv2026

Fibroblast-derived Collagen VI shapes the structure and function of the tumor-immune microenvironment in clear cell renal cell carcinoma

The importance of the extracellular matrix (ECM) influencing tumor biology in stroma-rich tumors is well established. However, the relevance of individual ECM proteins in rather stroma-poor cancers such as clear cell renal cell carcinoma (ccRCC) is ill-defined. Using bulk proteomics, spatial imaging, and single-cell transcriptomics, we identify collagen VI (COL6) as a predominant ECM component of the ccRCC interstitial stroma, synthesized primarily by fibroblasts and pericytes. Using cell-derived matrix (CDM) models, we demonstrate that COL6 is essential for maintaining an isotropic ECM network architecture and governs the broader matrisomal composition, with direct pro-proliferative consequences for tumor cells both in vitro and in situ. Granular spatial analysis reveals that COL6-rich stromal septa constrain tumor-infiltrating T cells to boundary zones, where CD8+PD1+ phenotypes predominate. Importantly, tyrosine kinase inhibition (TKI) with cabozantinib suppresses COL6 expression in fibroblasts in vitro and in ex vivo tumor models, mirroring COL6-depleted CDM phenotypes. Our findings establish COL6 as a central stromal regulator of ccRCC tumor biology and immune contexture, revealing ECM remodeling as an underappreciated mechanism of TKI action, with implications for combination immunotherapy strategies.

cancer biology↗

bioRxiv2026

Deep phenotyping of ATDC5-derived in vitro cartilage organoids

Cartilage is characterized by a highly specialized extracellular matrix (ECM) secreted by chondrocytes and limited self-regenerative capacity. In vivo investigations of chondrogenesis are limited by difficult and traumatic access, especially in humans. While it is known for decades that disturbances of chondrocyte differentiation and changed cartilage ECM composition cause severe skeletal phenotypes in vertebrates, a detailed molecular understanding of chondrogenesis and cartilage ECM formation is still missing, especially in the context of human genetic skeletal diseases. ATDC5 cells, derived from AT805 mouse teratocarcinoma cells, have been used in the past to model chondrogenic differentiation, however, most studies have investigated few major cellular differentiation markers only so that the composition of the secreted ECM as well as effects on the ATDC5 transcriptome upon differentiation are still unclear. Here, we performed time-resolved transcriptomic and ECM proteomic analyses of differentiating ATDC5 cells. Both datasets confirmed the formation of a cartilage-like matrix with increasing expression of key chondrocyte genes over the course of differentiation. ECM proteomics further revealed a number of ECM components not previously reported in ATDC5 cells or the secreted ECM, encompassing collagens, proteoglycans, glycoproteins and other secreted factors. Overall, our findings provide a more detailed molecular characterization of ATDC5 chondrogenesis and highlight the potential of this model system for ECM-focused studies.

cell biology↗

bioRxiv2025

MR-SP2: A Microreactor for Upward Pressure-Catapulting Laser Microdissection for Mass Spectrometry-Based Spatial Proteomics at Single-Cell Resolution

Laser capture microdissection (LCM) combined with liquid chromatography-tandem mass-spectrometry (LC-MS/MS) enables spatially resolved proteomics at few-cell scale, yet losses from minute LCM-cut specimens, particularly with upward pressure-catapulting systems and subsequent processing, limit depth and reproducibility. We present MR-SP{superscript 2} (Microreactor-based Sample Preparation for Spatial Proteomics), a one-pot-in-solution workflow that integrates reproducible LCM-cut specimen capture, processing with minimized adsorptive losses, and pipetting-free transfer with Evotip disposable precolumns. The workflow is demonstrated on a formalin-fixed paraffin-embedded (FFPE) murine kidney using upward pressure-catapulting LCM for targeted isolation of defined tissue specimens. Across 50,000 {micro}m3 regions (22 cells), MR-SP{superscript 2} modestly improved proteome coverage (3,381 {+/-} 80 versus 3,174 {+/-} 59 proteins). Decreasing sample input further accentuated the advantage of MR-SP{superscript 2} in maintaining higher identification rates, highlighting the successful reduction of adsorptive losses of the MR-SP{superscript 2} workflow. At 12,500 {micro}m3 (5-6 cells), identifications increased to 1,145 {+/-} 188 versus 302 {+/-} 126. At 3,125 {micro}m3 (1-2 cells), identifications reached 695 {+/-} 112 versus 206 {+/-} 51. MR-SP{superscript 2} improves identification depth for few-cell FFPE samples nearly threefold and provides an LCM-compatible preparation that expands the robustness, applicability of upward pressure-catapulting LCM within the spatial proteomics toolkit.

cancer biology↗

bioRxiv2025

Proteomic characterization of sporadic clear cell renal cell carcinoma reveals a matrix-dense pseudocapsule and heterogeneous tumor subtypes

Clear cell renal cell carcinoma (ccRCC) frequently develops a fibrotic pseudocapsule (PC) at the tumor boundary, yet its implications in the disease remain incompletely defined. We performed DIA-based proteomics on formalin-fixed, paraffin-embedded (FFPE) specimens of sporadic ccRCC from 153 patients, comprising 142 tumor, 123 pseudocapsule (PC), and 121 non-malignant adjacent tissue (NAT) samples. The PC displayed a distinctive proteomic signature characterized by matrix accumulation, enhanced remodeling and ECM-cell signaling, consistent with a signaling-competent boundary and active growth factor sequestration rather than a passive fibrotic barrier. Within the tumor, we observed canonical metabolic reprogramming with upregulated glycolysis and hypoxia markers and suppression of aerobic metabolism, accompanied by strong cell-cycle and pro-angiogenic signatures. Immune programs included increased antigen processing and presentation, together with elevated inflammasome and pyroptosis signatures. T-cell markers were enriched in the PC, indicating an immune-active boundary. Among tumors, we identified five distinct proteomic subtypes (C1-C5) spanning proliferative/mitotic, metabolic, immune/EMT/mTOR, and ECM phenotypes. Semi-specific peptide analysis indicated elevated endogenous proteolysis in the tumor and varied across clusters, with C1 and C5 showing the greatest proteolytic burden. Mechanotransduction features showed only modest PC elevation but were dominated by inter-patient variation. The present study defines a matrix-rich, signaling-active pseudocapsule and a classification of heterogeneous tumor subtypes in sporadic ccRCC. The framework provides a compartment-resolved, cluster-informed approach, and it highlights actionable axes of potential clinical relevance.

molecular biology↗

bioRxiv2025

In Situ, Antibody-Independent, and Multiplexed Characterization of Amyloid Plaques by MALDI MS/MS Imaging Using iprm-PASEF

BackgroundAmyloidosis collectively describes a heterogeneous group of protein aggregation-based diseases involving the misfolding and extracellular accumulation of fibril-forming amyloid proteins. The tissue deposition of these fibrils occurs either localized (limited to one organ) or systemically, impairing organ function and, in severe cases, leading to organ failure. Identification of the amyloidogenic protein leading to the correct subtyping is challenging but crucial for treatment decisions. Imaging parallel reaction monitoring (iprm)-parallel accumulation-serial fragmentation (PASEF) is a novel method for matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) that enables the fast, spatially resolved, multiplexed, and antibody-independent identification of peptides and proteins in situ. In this proof-of-concept study, we demonstrate the applicability of iprm-PASEF for the in situ characterization of amyloidosis. MethodsA multiplexed iprm-PASEF panel (comprising trapped ion mobility spectrometry (TIMS) and m/z values) for characterizing amyloid plaques by MALDI-MSI was compiled. The panel included nine peptide entries of amyloidosis-associated proteins (vitronectin, apolipoprotein E, serum amyloid P component) and the prevalent amyloidosis-subtype proteins serum amyloid A, transthyretin, and immunoglobulin light chain 2. The multiplexed iprm-PASEF panel was applied to a tissue microarray (TMA) assembled from formalin-fixed, paraffin-embedded (FFPE) specimens, comprising biopsies of amyloidosis-positive tissues from 18 patients, including six different tissue types, and representing the following amyloidosis subtypes: ATTR (transthyretin amyloidosis), AL (immunoglobulin light chain amyloidosis), and AA (secondary amyloidosis). For comparison and co-localization, congo red staining was performed on adjacent slides. ResultsMALDI TIMS MS1 imaging was acquired from the amyloidosis TMA, providing an m/z feature list containing 1390 entries that was subsequently refined to only include m/z features likely stemming from amyloidosis-related peptides. Further, we focused on optimal usage of the m/z and ion mobility range to increase the number of targeted peptides. The final iprm-PASEF panel comprised 10 entries ranging from m/z 887.51 - 1,986.85 and 1/K0 1.4 - 2.1 V{middle dot}s/cm2; targeting nine peptides from six different amyloidosis-related proteins. Apolipoprotein E, serum amyloid P component, and immunoglobulin light chain 1/2 were included with two entries. Transthyretin, vitronectin, and serum amyloid A were covered by one peptide. One entry was included as a positive control targeting actin A. We applied the iprm-PASEF (MS2 mode) panel to the amyloidosis TMA and fragment (MS2) spectra were analyzed by the search engine MASCOT. Eight out of ten peptides derived from vitronectin, apolipoprotein E, serum amyloid A, serum amyloid P component, and transthyretin were successfully identified with MASCOT scores above 18. Comparison to Congo red staining indicated a more localized and heterogeneous pattern of amyloid-associated proteins (vitronectin, apolipoprotein E, serum amyloid P component) by iprm-PASEF. Compared to the clinical annotation, transthyretin and serum amyloid A were only found in their respective subtypes. In total, it was possible to describe the spatial distribution of five different amyloid-associated proteins, thereby further characterizing Congo red-positive amyloid plaques in one single iprm-PASEF measurement. Conclusion and perspectiveThis proof-of-concept study demonstrates feasibility for in situ, antibody-independent, and multiplexed proteomic typing of amyloidosis plaques. Future enlargement of the multiplex panel is likely to increase the number of targetable amyloid proteins and to probe for the presence of post-translational modifications. The present study highlights the value of iprm-PASEF for MALDI imaging strategies.

molecular biology↗

bioRxiv2025

Chlamydial membrane vesicles deliver the chlamydial porin OmpA to mitochondria to inhibit apoptosis

Chlamydiae are obligate intracellular bacteria that inhibit mitochondrial apoptosis to maintain integrity of the host cell. We have previously reported that a chlamydial outer membrane {beta}-barrel protein, the porin OmpA, can during ectopic expression inhibit mitochondrial apoptosis through direct interaction with the BCL-2-family effectors BAX and BAK. We here show that OmpA from Chlamydia trachomatis (Ctr) uses membrane vesicles for its delivery to the outer mitochondrial membrane during Ctr infection. Using a number of imaging and fractionation techniques, we show that OmpA during infection reaches mitochondria and is inserted into mitochondrial membranes. Purified membrane vesicles (MV) from Ctr-infected cells contained OmpA. When added to uninfected cells, MV fused with mitochondrial membranes, causing the interaction of OmpA with BAK and the cytosolic retro-translocation of BAX. MV addition to uninfected cells protected the cells against apoptosis. We propose a structural model of this BAK inhibition by OmpA that reenacts the inhibition of BAK by the mitochondrial porin VDAC2. The results provide evidence that the porin from Chlamydia, as well as the structurally similar porin from the related Simkania, specifically exploits its relationship to mitochondrial porins to protect the infected cell against apoptosis and to enable intracellular growth of the bacteria in human cells.

cell biology↗

bioRxiv2025

Specificity profiling of SARS-CoV-2 PLpro using proteome-derived libraries of linear peptides suggests secondary preference for basic motifs

SARS-CoV-2 papain-like protease (PLpro) is essential for viral replication and immune modulation. Here, a proteomic identification of protease cleavage sites (PICS) approach was applied using proteome-derived peptide libraries to determine the enzymes substrate specificity. PLpro exhibited a yet unreported preference for basic amino acids at P1, primarily arginine or lysine. This secondary specificity frequently involved cleavage between two basic residues (e.g., K|K, K|R or R|K). Experiments with in-house and commercial PLpro in GluC-generated peptide libraries from Escherichia coli and HEK293 proteomes confirmed this preference, though with lower overall efficiency compared to typical trypsin-like proteases. SARS-CoV-1 PLpro likewise displayed this basic-site specificity, underscoring its conservation across related coronaviruses. Site-directed mutagenesis of acidic residues near the catalytic triad to neutral variants altered cleavage preferences, indicating the involvement of these side chains in substrate binding and potential alternative binding modes. We also evaluated wild-type PLpro specificity on intact protein lysates rather than peptide libraries to assess how structure influences cleavage patterns. Notably, P1 arginine specificity persisted at the protein level, whereas lysine specificity was reduced, suggesting additional structural constraints in complex substrates. A strong presence of glycine on the prime side further suggests a bias toward unstructured regions. These findings reveal an expanded substrate recognition repertoire for SARS-CoV-2 PLpro, which may be relevant for the design of targeted inhibitors and understanding of viral protease function.

biochemistry↗

bioRxiv2025

Vertical RAS-pathway inhibition in pancreatic cancer drives therapeutically exploitable mitochondrial alterations

Background & AimsOncogenic KRAS mutations drive metabolic rewiring in pancreatic ductal adenocarcinoma (PDAC). Src-homology 2 domain-containing phosphatase 2 (SHP2) is essential for full KRAS activity and promising dual SHP2/mitogen-activated protein kinase (MAPK) inhibition is currently being tested in clinical trials. Exploitable metabolic adaptations may contribute to an invariably evolving resistance. MethodsTo understand the metabolic changes induced by dual inhibition, we comprehensively tested cell lines, endogenous tumor models, and patient-derived organoids representing the full spectrum of PDAC molecular subtypes. ResultsWe find that dual SHP2/mitogen-activated protein kinase kinase (MEK1/2) inhibition induces major mitochondrial alterations, elevates reactive oxygen species (ROS) levels and triggers a lipid peroxidase dependency. While anabolic pathways, glycolysis and autophagy were also affected, mitochondrial alterations persisted longterm into a therapy resistant state. ConclusionsThe resulting vulnerability to induction of ferroptotic cell death via combined SHP2/MEK1/2 and glutathione peroxidase (GPX4) inhibition provides a metabolic lever to reinforce RAS-pathway inhibition for targeted PDAC treatment.

cancer biology↗

bioRxiv2024

Spatial Proteomics by Trapped Ion Mobility supported MALDI MS/MS Imaging: A First Glance into Multiplexed and Spatial Peptide Identification

RATIONALEIn spatial proteomics, matrix-assisted laser desorption/ionization (MALDI) imaging enables rapid and cost-effective peptide measurements. Yet, in situ peptide identification remains challenging. Therefore, this study aims to integrate the trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) into MALDI imaging of tryptic peptides to enable multiplexed MS/MS imaging. METHODSAn initial MALDI TIMS MS1 survey measurement was performed, followed by a manual generation of a precursor list containing mass over charge values and ion mobility windows. Inside the dual TIMS system, submitted precursors were trapped, separately eluted by their ion mobility and analyzed in a quadrupole time-of-flight device, thereby enabling multiplexed MALDI MS/MS imaging. Finally, precursors were identified by peptide to spectrum matching. RESULTSThis study presents the first multiplexed MALDI TIMS MS/MS imaging (iprm-PASEF) of tryptic peptides. Its applicability was showcased on two histomorphologically distinct tissue specimens in a 4-plex and 5-plex setup. Precursors were successfully identified by the search engine MASCOT in one single MALDI imaging experiment for each respective tissue. Peptide identifications were corroborated by liquid-chromatography tandem mass spectrometry experiments and fragment co-localization analyses. CONCLUSIONSIn this study, we present a novel pipeline, based on iprm-PASEF that allows the multiplexed and spatial identification of tryptic peptides in MALDI imaging. Hence, it marks a first step towards the integration of MALDI imaging into the emerging field of spatial proteomics.

biochemistry↗

bioRxiv2024

The RRM domain-containing protein Rbp3 interacts with ribosomes and the 3' ends of mRNAs encoding photosynthesis proteins

RNA recognition motif (RRM) domain proteins are crucial RNA-binding proteins (RBPs) across all domains of life. In cyanobacteria, single RRM domain proteins are involved in mRNA targeting to the thylakoid membrane and acclimation to certain stress conditions, but many details of their physiological functions and molecular targets have remained unknown. The model cyanobacterium Synechocystis sp. PCC 6803 has a family of three genes encoding the RRM domain-containing proteins Rbp1, Rbp2 and Rbp3. Here, we verified the RNA-binding activity of Rbp3 in vivo and show that cells of a {Delta}rbp3 deletion strain had a lower PSI:PSII ratio and decreased pigment content and were significantly smaller than wild-type cells. To identify the set of interacting molecules, co-immunoprecipitation experiments were performed with a strain expressing a C-terminally FLAG-tagged Rbp3. Mass spectrometry of the elution fraction suggested physical proximity between Rbp3, ribosomes, and a very small number of other proteins. The most highly enriched transcript in the co-eluting RNA fraction was the psaAB mRNA. This was corroborated by fluorescent in situ hybridization (FISH) analyses showing decreased psaA mRNA signals in {Delta}rbp3, and colocalization with Rbp3-GFP in the wild type. Other mRNAs co-enriched with Rbp3 encode thylakoid, plasma membrane and carboxysome proteins. Binding assays using Bio-layer Interferometry validated the Rbp3-psaAB mRNA interaction, indicating a preference for folded RNA segments near or overlapping the respective stop codons. Significance statementThe mechanisms by which proteins are produced at specific sites and inserted into the intricate membrane systems of photosynthetic cyanobacteria are only partially understood. While RRM domain proteins are well-studied RNA-binding proteins in eukaryotes, their functions in bacteria remain underexplored. This study reveals that the RRM domain protein Rbp3 in the cyanobacterium Synechocystis sp. PCC 6803 binds mRNAs encoding photosynthetic proteins, plasma membrane proteins and carboxysome proteins and localizes near ribosomes. The bound RNA segments are typically near the ends of coding regions, or in 5' untranslated regions. These findings suggest that Rbp3 is involved in targeting mRNAs to various intracellular locations by interacting with structural elements within these mRNA molecules.

microbiology↗

bioRxiv2024

Versatile roles of Annexin A4 in ccRCC: impact on membrane repair, transcriptional signatures, and composition of the tumor microenvironment

Clear cell renal cell carcinoma (ccRCC) is the most prevalent type of renal malignant disease and is characterized by dismal prognosis in the metastasized setting. Invasive growth of cancer cells relates to high levels of compressive forces translating to relevant damage of the plasma membrane. However, functional implications of protein machineries required for plasma membrane repair in ccRCC are not yet completely elucidated. Given the membrane-associated localization of the large family of annexin proteins, we aimed for a global annotation of annexin proteins, which led to the identification of ANXA4 selectively expressed in cancer cells of ccRCC. Interestingly, ANXA4 showed context-dependent distinct localization patterns including the plasma membrane as well as the nuclear compartment/nuclear membrane. We investigated the functional role of ANXA4 in ccRCC employing genetic titration studies (knockdown, CRISPR/Cas9 knockout and overexpression) and identified impaired acute plasma membrane repair as well as invasive capability in conditions of reduced ANXA4 expression. Utilizing computational segmentation of the tumor microenvironment (TME) of ccRCC samples revealed that ANXA4 low tumors exhibited a distinct TME composition compared to ANXA4 high cases. ANXA4 low tumors showed higher levels of tumor infiltrating lymphocytes accompanied by increased deposition of acellular extracellular matrix. Further transcriptomic analysis demonstrated major alterations in transcriptional signatures related to epithelial-mesenchymal transition (EMT) and immune signaling. Transcription factor enrichment analysis and further functional validation identified ELF3 as one central regulator of invasive properties. Our integrative approach including molecular analyses with advanced histopathological segmentation uncovered novel roles for ANXA4 in modulating acute membrane repair, transcriptional regulation, and shaping cellular composition of the ccRCC tumor microenvironment.

cancer biology↗

bioRxiv2024

Serum Proteome Profiling Identifies N-Cadherin and C-Met as Early Marker Candidates of Therapeutic Response to Neoadjuvant Chemotherapy in Breast Cancer

Breast cancer remains the most common cancer in women worldwide. Neoadjuvant chemotherapy (NACT) is often preferred to adjuvant chemotherapy to achieve tumour shrinkage, monitor response to therapy and facilitate surgical removal in the absence of metastases. In addition, there is strong evidence that pathological complete remission (pCR) is associated with prolonged survival. In this study, we sought to identify candidate markers that signal response or resistance to therapy. We present a retrospective longitudinal serum proteomic study of 22 breast cancer patients (11 with pCR and 11 with non-pCR) matched with 21 healthy controls. Serum was analysed by LC-MS/MS after depletion of abundant proteins by immunoaffinity, trypsinisation, isobaric labelling and fractionation by reversed-phase HPLC. We observed an inverse behaviour of the serum proteins c-Met and N-cadherin after the second cycle of chemotherapy with a high predictive value (AUC 0.93). More pronounced changes were observed after the 6th cycle of NACT, with significant changes in the intensity of the proteins contactin-1, centrosomal protein, sex hormone-binding globuline and cholinesterase. Our study highlights the possibility of monitoring response to NACT using serum as a liquid biopsy.

cancer biology↗

bioRxiv2024

Proteomic profiling of IDH-wildtype Glioblastoma Tissue and Serum uncovers prognostic Subtypes and Marker Candidates

BackgroundIDH-wildtype glioblastoma (GBM) is the most prevalent primary brain cancer with a 5-year survival rate below 10%. Despite combined treatment through extensive resection and radiochemotherapy, nine out of ten patients develop recurrences. The lack of targeted treatment options and reliable diagnostic markers for recurrent tumors remain major challenges. Methods & AimsIn this study, we present the proteomic characterization of tissue and serum from 55 initial GBM tumors and five matching recurrences, which we investigated for proteomic tumor subtypes and proteomic signatures associated with recurrence. ResultsPrimary tumors revealed four distinct subgroups through hierarchical clustering: a neuronal cluster with elevated mature neuron markers, an innate immunity cluster with increased protease expression, a mixed cluster, and a stem-cell cluster. Neurodevelopmental and inflammatory processes were identified as key factors influencing clustering, with proteolytic activity increasing relative to the degree of inflammation. An analysis comprising proteins with lower coverage confirmed and expanded this pattern. Patients in the neuronal cluster exhibited significantly longer survival compared to those in the stem-cell cluster. In a patient-matched differential expression analysis, five recurrent tumors displayed significantly altered protein expression compared to their primary counterparts, emphasizing the proteomic plasticity of recurrent tumors. Investigation of serum proteomes before and after surgery, using a depletion-based protocol, revealed highly patient-specific and stable proteome compositions, despite a notable increase in inflammation markers post-surgery. However, the levels of circulating proteolytic products matched to the proteolytic activity within the tissue and one fragment of proteolysis activated receptor 2 (PAR2) consistently dropped in abundance after removal of inflamed tumors. ConclusionOverall, we describe a large proteomic GBM cohort. We identified distinct tumor subgroups, molecular patterns of recurrence, and matching proteomic patterns in the bloodstream, which may improve risk prediction for recurrent GBM.

cancer biology↗

bioRxiv2024

The Role of the Co-Chaperone DNAJB11 in Polycystic Kidney Disease: Molecular Mechanisms and Cellular Origin of Cyst Formation

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), which are required for the regulation of the renal tubular diameter. Loss of polycystin function results in cyst formation. Atypical forms of ADPKD are caused by mutations in genes encoding endoplasmic reticulum (ER)-resident proteins through mechanisms that are not well understood. Here, we investigate the function of DNAJB11, an ER co-chaperone associated with atypical ADPKD. We generated mouse models with constitutive and conditional Dnajb11 inactivation and Dnajb11-deficient renal epithelial cells to investigate the mechanism underlying autosomal dominant inheritance, the specific cell types driving cyst formation, and molecular mechanisms underlying DNAJB11-dependent polycystic kidney disease. We show that biallelic loss of Dnajb11 causes cystic kidney disease and fibrosis, mirroring human disease characteristics. In contrast to classical ADPKD, cysts predominantly originate from proximal tubules. Cyst formation begins in utero and the timing of Dnajb11 inactivation strongly influences disease severity. Furthermore, we identify impaired PC1 cleavage as a potential mechanism underlying DNAJB11-dependent cyst formation. Proteomic analysis of Dnajb11- and Pkd1-deficient cells reveals common and distinct pathways and dysregulated proteins, providing a foundation to better understand phenotypic differences between different forms of ADPKD.

molecular biology↗

bioRxiv2024

Proteomic Characterization of Intrahepatic Cholangiocarcinoma Identifies Distinct Subgroups and Proteins Associated with Time-To-Recurrence

Background & AimsIntrahepatic cholangiocarcinoma (ICC) is a poorly understood cancer with dismal survival and high recurrence rates. ICCs are often detected in advanced stages. Surgical resection is the most important first-line treatment but limited to non-advanced cases, whereas chemotherapy provides only a moderate benefit. The proteome biology of ICC has only been scarcely studied and the prognostic value of initial ICCs proteomic features for the time-to-recurrence (TTR) remains unclear. MethodsWe dissected formalin-fixed, paraffin-embedded samples from 80 tumor- and 77 matching adjacent non-malignant (TANM) tissues. All samples were measured via liquid-chromatography mass-spectrometry (LC-MS/MS) in data independent acquisition mode (DIA). ResultsTumor- and TANM tissue showed strongly different biologies and DNA-repair, translation, and matrisomal processes were upregulated in ICC. In a hierarchical clustering analysis, we determined two proteomic subgroups of ICC, which showed significantly diverging TTRs. Cluster 1, which is associated with a beneficial prognosis, was enriched for matrisomal processes and proteolytic processing, while cluster 2 showed increased RNA and protein turnover. In a second, independent Cox proportional hazards model analysis, we identified individual proteins whose expression correlates with TTR distribution. Proteins with a positive hazard ratio were mainly involved in carbon/glucose metabolism and protein turnover. Conversely, proteins associated with a low hazard ratio were mostly linked to the extracellular matrix. Additional proteome profiling of patient-derived xenograft tumor models of ICC successfully distinguished tumor and stromal proteins and provided insights into cell-matrix interactions. ConclusionsWe successfully determine the proteome biology of ICC and present two proteome clusters in ICC patients with significantly different TTR rates and distinct biological motifs. A xenograft model confirmed the importance of tumor-stroma interactions for this cancer.

cancer biology↗

bioRxiv2024

Characterization of YlxR/Ssr1238, a conserved RNA binding protein in a model cyanobacterium

Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Structural prediction suggests an RNA-binding interface for the protein YlxR/Ssr1238 in the cyanobacterium Synechocystis 6803. Two pairs of cysteine residues are arranged as possibly coordinating an Fe-S cluster and appear widely conserved in the homologous proteins of other cyanobacteria. Overexpression of Ssr1238 for 24 h led to higher levels of RNase P RNA, tRNAs, and stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins. A second highly enriched transcript derived from gene ssl3177, which encodes a central enzyme in cell wall remodeling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA modifying enzymes, riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. In conclusion, Ssr1238 specifically binds to two different transcripts and participates in the coordination of RNA maturation, translation, cell division, and aspects of nitrogen metabolism. Our results are consistent with recent findings that the B. subtilis YlxR protein functions as an RNase P modulator (RnpM), but suggest additional functionalities and extend its proposed role to the phylum cyanobacteria.

microbiology↗

bioRxiv2023

Alternative mRNA splicing controls the functions of the histone H3K27 demethylase UTX/KDM6A

BackgroundThe UTX/KDM6A histone H3K27 demethylase plays an important role in development and is frequently mutated in cancers such as urothelial cancer. Despite many studies on UTX proteins, variations in mRNA splicing have been overlooked. MethodsUsing Nanopore sequencing, we present a comprehensive analysis of UTX/KDM6A splicing events in human cell lines and in tissue samples from bladder cancer and normal epithelium. ResultsThe central region of UTX mRNAs encoded by exons 12 to 17 undergoes extensive alternative splicing. Up to half of all stable mRNAs (8-48% in bladder tissues and 18-58% in cell lines) are represented by the UTX canonical isoform lacking exon 14 encoding a nuclear localization sequence, hence localize to the nucleus, unlike cytonuclear localization of the canonical isoform. Chromatin association was also higher for exon 14-containing isoform compared to the canonical UTX. Using quantitative mass spectrometry, we found that all UTX isoforms integrated into the MLL3 and MLL4, PR-DUB and MiDAC complexes. Interestingly, one of the novel UTX isoforms, which lacks exons 14 and 16, fails to interact with PR-DUB and MiDAC complex members. ConclusionUTX mRNAs undergo extensive alternative splicing that controls the subcellular localization of UTX and its interactions with other chromatin regulatory complexes. Simple SummaryUTX/KDM6A is a histone H3K27 demethylase and plays an important role in mammalian development and human diseases such as urothelial cancer. We identified a region encompassing exons 12-17 of UTX that undergoes extensive splicing events. As a result, a nuclear localization sequence located in exon14 is missing in a considerable part of UTX transcripts in different cell lines and tissues from normal bladder epithelium and bladder cancer. Mass spectrometry analysis showed a role for this region in binding to the epigenetic PR-DUB and MiDAC complexes. UTX was also more extensively bound to chromatin when the alternative splicing region presented. Our study showed that alternative splicing of UTX transcripts plays an important role in its functions.

biochemistry↗