Biology subjects

Schlosser, A.

Publications and source records attributed to Schlosser, A..

15 recordsLinked to original sources

bioRxiv2026

Robust ORF Deconvolution from Multiple Ribo-seq Datasets with Group LASSO Penalty

Ribo-seq has provided compelling evidence that there are many translated but unannotated ORFs. Despite a growing body of peptidomic support for cryptic ORFs, the computational discovery of weakly translated, short and mutually overlapping ORFs has remained a major bottleneck. Here we introduce PRICE 2, a method designed to resolve these challenging translation events by adapting concepts from isoform deconvolution. PRICE 2 employs a generative model combined with group LASSO regularized regression to jointly infer active ORFs across multiple sequencing runs. In a comprehensive benchmark, PRICE 2 outperformed thirteen state-of-the-art tools in detecting short and overlapping ORFs. Orthogonal validation via immunopeptidomics demonstrated that ORFs identified by PRICE 2 enable the discovery of [~]40% more cryptic MHC-I peptides than the best-performing competing tool at a fixed false discovery rate. By analyzing public ribo-seq data across 45 distinct cell and tissue types, we constructed ORF-stock, a comprehensive database of non-canonical ORFs for immunopeptidomic studies.

bioinformatics↗

bioRxiv2025

Axon guidance deficits in a human sensory neuron model of Fabry disease

Fabry disease (FD) is a rare genetic galactosidase alpha (GLA) gene associated lysosomal disorder caused by alpha-galactosidase A (AGAL) deficiency, leading to sphingolipid (globotriaosylceramide, Gb3) accumulation in multiple tissues. Burning pain due to small fiber neuropathy is an early symptom with great impact on health- related quality of life. The pathophysiological role of Gb3 accumulations in sensory neurons of the dorsal root ganglia is incompletely understood. We have differentiated induced pluripotent stem cells of an isogenic GLA knockout line (p.S364del, hemizygous) and its healthy control into sensory neurons to model FD in vitro. We have compared both lines on transcriptional and proteomic level and investigated the effects of AGAL enzyme supplementation. FD sensory neurons showed dysregulation of disease-related pathways, including axon guidance at both RNA and protein level and microfluidic assays revealed shorter neurite length. While AGAL did not restore the transcriptomic state, it reduced Gb3 accumulation and lowered protein ephrin 5A and glycoprotein M6A level. These findings highlight axon guidance alterations in an isogenic human FD sensory model, with potential implications for early central and peripheral innervation in small fiber neuropathy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/673441v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@199cd19org.highwire.dtl.DTLVardef@682521org.highwire.dtl.DTLVardef@15da60borg.highwire.dtl.DTLVardef@1404c10_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Murine cytomegalovirus evolved a cell-cycle regulator (m54.5) within the highly conserved viral DNA polymerase gene

Ribosome profiling (Ribo-seq) coupled with transcription start site profiling time-course analyses recently unveiled hundreds of novel viral gene products in lytic murine cytomegalovirus (MCMV) infection. One of these is the m54.5 open reading frame (ORF) located within the highly conserved viral DNA polymerase locus (M54). Interestingly, the m54.5 ORF is expressed from its own transcript (m54.5 RNA) with early gene expression kinetics, and at much higher levels than M54. In this study, we show that m54.5 encodes a nuclear viral protein (m54.5p) that contributes to cell cycle regulation during lytic MCMV infection. We show that m54.5p interacts with components of the anaphase-promoting complex/cyclosome (APC/C) and the phosphatase-6 (PP6) complex. Nocodazole mitotic arrest assays confirmed G1 cell cycle arrest and dysregulation by m54.5. Serum starvation revealed impaired cell cycle progression to S-phase. Notably, m54.5p is not conserved in other cytomegaloviruses but functionally mimics the UL21a protein of human cytomegalovirus (HCMV), which similarly targets the master cell cycle regulator APC/C to disrupt cell cycle progression. m54.5 thus represents convergent evolution to HCMV UL21a in MCMV within the highly conserved viral DNA polymerase gene. Nevertheless, we found that m54.5p is dispensable for viral replication in cultured mouse fibroblasts, indicative of redundant cell cycle regulation in lytic MCMV infection. These findings highlight a surprising genomic plasticity of herpesviruses, facilitating the evolution of an independent transcript encoding for a >200 aa gene product within a deeply conserved viral gene locus. Author SummarySystems biology approaches have revealed a surprising complexity of herpesvirus gene products. Using advanced sequencing approaches, we discovered a novel gene, m54.5, that independently evolved within a highly conserved region of the murine cytomegalovirus (MCMV) genome. This gene, which shows no conservation in other CMVs, produces a nuclear protein, m54.5p, abundantly expressed early during infection. We show that m54.5p interacts with host cell cycle regulators--the anaphase-promoting complex/cyclosome (APC/C) and phosphatase-6 (PP6)--to arrest cells in G1 phase and block progression into S phase. This function and underlying mechanism are reminiscent of the unrelated UL21a protein in human cytomegalovirus, illustrating how distinct viruses can evolve similar strategies to control host cell division. Despite its role in cell cycle disruption, m54.5p is not required for MCMV replication in cultured cells, suggesting redundant viral mechanisms. Our findings reveal an unexpected plasticity of herpesvirus genomes to evolve new, functional transcripts and proteins even within one of the most highly conserved genomic regions. Our findings thereby reshape our understanding of herpesvirus evolution and virus-host interaction.

microbiology↗

bioRxiv2025

Stable interaction of NONO with DBHS family members upon etoposide-induced DNA damage

The Non-POU domain containing octamer binding (NONO) protein is a member of the multifunctional Drosophila behavior/human splicing (DBHS) protein family and a core component of nuclear paraspeckles. NONO forms dimers with the other two DBHS members splicing factor proline and glutamine rich (SFPQ) protein or the paraspeckle component 1 (PSCP1) to modulate RNA metabolism and gene expression both at the transcriptional and post-transcriptional level. Increasing evidence suggests that NONO participates in genome maintenance by stimulating the DNA damage response (DDR) upon induction of DNA double-strand breaks (DSBs). However, the molecular principles that engage NONO in genome stability are poorly understood. We hypothesized that the induction of DSBs alters NONO protein-protein interactions and applied label-free mass spectrometry to test for changes in the interactome of NONO in human U2OS cells upon treatment with the topoisomerase II inhibitor etoposide. Surprisingly, our mass spectrometry data reveal that etoposide treatment does not induce major changes in NONO protein-protein interactions. We confirmed this finding by orthogonal co-immunoprecipitation assays and co-localization assays. Our data suggest that the bulk of interactions between NONO and SFPQ or PSPC1 are insensitive to etoposide treatment and that DBHS family members promote genome stability as stable dimers.

biochemistry↗

bioRxiv2024

SILAC-based quantification reveals modulation of the immunopeptidome in BRAF and MEK inhibitor sensitive and resistant tumor cells

The immunopeptidome is constantly monitored by T cells to detect foreign or aberrant HLA peptides. It is highly dynamic and reflects the current cellular state, enabling the immune system to recognize abnormal cellular conditions, such as those present in cancer cells. To precisely determine how changes in cellular processes, such as those induced by drug treatment, affect the immunopeptidome, quantitative immunopeptidomics approaches are essential. To meet this need, we developed a pulsed SILAC-based method for quantitative immunopeptidomics. Metabolic labeling with lysine, arginine, and leucine enabled isotopic labeling of nearly all HLA peptides across all allotypes (> 90% on average). We established a data analysis workflow that integrates the de novo sequencing-based tool Peptide-PRISM for comprehensive HLA peptide identification with MaxQuant for accurate quantification. We employed this strategy to explore the modulation of the immunopeptidome upon MAPK pathway inhibition (MAPKi) and to investigate alterations associated with early cellular responses to inhibitor treatment and acquired resistance to MAPKi. Our analyses demonstrated significant changes in the immunopeptidome early during MAPKi treatment and in the resistant state. Moreover, we identified putative tumor-specific cryptic HLA peptides linked to these processes that might represent exploitable targets for cancer immunotherapy. SynopsisWe have developed a mass spectrometric method that allowed us to investigate the effects of common MAPK inhibitors on the immunopeptidome of melanoma cells. This approach has led to the discovery of new potential targets for cancer immunotherapy.

immunology↗

bioRxiv2024

A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins

Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. Additionally, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins. Key messageAnalysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear encoded, mitochondria-localized proteins. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/598444v3_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f31702org.highwire.dtl.DTLVardef@6b9ce6org.highwire.dtl.DTLVardef@109c041org.highwire.dtl.DTLVardef@12cf83e_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

Peptide-stimulation of autologous T cells reverts immune checkpoint inhibitor resistance of hypermutated colorectal cancer cells

Two hypermutated colon cancer cases with patient-derived cell lines, peripheral and tumor-infiltrating T cells available were selected for detailed investigation of immunological response. T cells co-cultured with autologous tumor cells showed only low levels of pro-inflammatory cytokines and failed at tumor recognition. Similarly, treatment of co-cultures with immune checkpoint inhibitors (ICI) did not boost antitumor immune responses. Since proteinase inhibitor 9 (PI-9) was detected in tumor cells, a specific inhibitor (PI-9i) was used in addition to ICI in T cell cytotoxicity testing. However, only pre-stimulation with tumor-specific peptides (cryptic and neoantigenic) significantly increased recognition and elimination of tumor cells by T cells independently of ICI or PI-9i. We showed, that ICI resistant tumor cells can be targeted by tumor-primed T cells and also demonstrated the superiority of tumor-naive peripheral blood T cells compared to highly exhausted tumor-infiltrating T cells. Future precision immunotherapeutic approaches should include multimodal strategies to successfully induce durable anti-tumor immune responses.

cancer biology↗

bioRxiv2023

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neurosecretory protein in the Drosophila CNS.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analysed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk/scRNA-seq and protein datasets from larval brains in which Alk signaling was manipulated, identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (Clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

developmental biology↗

bioRxiv2022

Immunogenicity of non-canonical HLA-tumor ligands identified through proteogenomics

Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the immunogenicity of nonC tumor HLA-I ligands (nonC-TL) to better understand their contribution to cancer immunosurveillance and their therapeutic applicability. Using proteogenomics, we identified 517 nonC-TL from 9 patients with melanoma, gynecological, and head and neck cancer. We found no recognition of the 507 nonC-TL tested by autologous ex vivo expanded tumor reactive T-cell cultures while the same cultures demonstrated reactivity to mutated, cancer-germline, or melanocyte differentiation antigens. However, in vitro sensitization of donor peripheral blood lymphocytes against 170 selected nonC-TL, led to the identification of T-cell receptors (TCRs) specific to three nonC-TL, two of which mapped to the 5 UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of C5orf22C. T cells targeting these nonC-TL recognized cancer cell lines naturally presenting their corresponding antigens. Expression of the three immunogenic nonC-TL was shared across tumor types and barely or not detected in normal cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they may be attractive novel targets for widely applicable immunotherapies.

cancer biology↗

bioRxiv2021

Sylites: Multipurpose markers for the visualization of inhibitory synapses

We introduce Sylites - small and versatile fluorogenic affinity probes for high-contrast visualization of inhibitory synapses. Having stoichiometric labeling and exceptional selectivity for neuronal gephyrin, a hallmark protein of the inhibitory post-synapse, Sylites enable superior synapse staining compared with antibodies. Combined with super-resolution microscopy, Sylites allow precise nanoscopic measurements of the synapse. In brain tissue, Sylites reveal the three-dimensional distribution of inhibitory synapses within just an hour.

neuroscience↗

bioRxiv2020

Structural basis for the activation of CDK7 by MAT1 and Cyclin H

CDK7, Cyclin H, and MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex. CAK is a vital factor for the two essential processes of transcription and cell cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH it phosphorylates the T-loop of CDKs that control cell cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on the MAT1 protein for activation in addition to Cyclin H. We solved the structure of the CAK complex from the model organism C. thermophilum at 2.6 [A] resolution. Our structure reveals an intricate network of interactions between MAT1 and its two binding partners CDK7 and Cyclin H providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements combined with functional mutagenesis show that CDK7 activation can occur independently of T-loop phosphorylation and is thus exclusively MAT1 dependent by positioning the CDK7 T-loop in its active conformation. Finally, our structure of the active CAK with a peptide model provides a molecular rationale for heptad repeat phosphorylation. Significance StatementThe fundamental processes of cell cycle regulation and transcription are linked by the heterotrimeric CDK-activating kinase (CAK) complex. We have solved the crystal structure of the active CAK complex and provide a molecular rationale for CAK activation, regulation, and substrate recognition thereby highly advancing our understanding of this essential factor.

biochemistry↗

bioRxiv2020

A phosphoproteomic approach reveals that PKD3 controls phenylalanine and tyrosine metabolism

Members of the Protein Kinase D (PKD) family (PKD1, 2, and 3) integrate hormonal and nutritional inputs to regulate complex cellular metabolism. Despite the fact that a number of functions have been annotated to particular PKDs, their molecular targets are relatively poorly explored. PKD3 promotes insulin sensitivity and suppresses lipogenesis in the liver. However, its substrates are largely unknown. Here we applied proteomic approaches to determine PKD3 targets. We identified over three-hundred putative targets of PKD3. Among them phenylalanine hydroxylase (PAH). PAH catalyses the conversion of phenylalanine to tyrosine and its activity is regulated by, phenylalanine concentration and glucagon-induced signaling. Consistently, we showed that PKD3 is activated by glucagon and promotes tyrosine levels in primary hepatocytes and liver of mice. Taken together, our comprehensive proteomic approach established that PKD3 determine the rate of phenylalanine to tyrosine conversion in the liver. Therefore, our data indicate that PKD3 might play a role in development of diseases related to the defective tyrosine and phenylalanine metabolism.

biochemistry↗

bioRxiv2020

CK2 inhibits TIMELESS nuclear export and modulates CLOCK transcriptional activity to regulate circadian rhythms

Circadian clocks orchestrate daily rhythms in organismal physiology and behavior to promote optimal performance and fitness. In Drosophila, key pacemaker proteins PERIOD (PER) and TIMELESS (TIM) are progressively phosphorylated to perform phase-specific functions. Whereas PER phosphorylation has been extensively studied, systematic analysis of site-specific TIM phosphorylation is lacking. Here, we identified phosphorylation sites of PER-bound TIM by mass spectrometry, given the importance of TIM as a modulator of PER function in the oscillator. Among the twelve TIM phosphorylation sites we identified, at least two of them are critical for circadian timekeeping as mutants expressing non-phosphorylatable mutations exhibit altered behavioral rhythms. In particular, we observed that CK2-dependent phosphorylation of TIM(S1404) promotes nuclear accumulation of PER-TIM heterodimers by inhibiting the interaction of TIM and nuclear export component, Exportin 1 (XPO1). We postulate that proper level of nuclear PER-TIM accumulation is necessary to facilitate kinase recruitment for the regulation of daily phosphorylation rhythm and phase-specific transcriptional activity of CLOCK (CLK). Our results highlight the contribution of phosphorylation-dependent nuclear export of PER-TIM heterodimers to the maintenance of circadian periodicity and identify a new mechanism by which the negative elements of the circadian oscillator (PER-TIM) regulate the positive elements (CLK-CYC). Finally, since the molecular phenotype of tim(S1404A) non-phosphorylatable mutant exhibits remarkable similarity to that of a mutation in human timeless that underlies Familial Advanced Sleep Phase Syndrome (FASPS), our results revealed an unexpected parallel between the functions of Drosophila and human TIM and may provide new insights into the molecular mechanisms underlying human FASPS. Significance StatementPhosphorylation is a central mechanism important for the regulation of circadian physiology across organisms. The molecular oscillator is composed of pacemaker proteins that undergo elaborate phosphorylation programs to regulate phase-specific functions. In Drosophila, phosphorylation of TIMELESS (TIM) has been recognized as critical for its function in the oscillator, but a systematic analysis of TIM phosphorylation is lacking. Here, we identified twelve Drosophila TIM phosphorylation sites by mass spectrometry and showed that phosphorylation at TIM(S1404) is necessary for maintaining 24-hour rhythms. Finally, since the molecular phenotype of tim(S1404A) non-phosphorylatable fly mutant exhibits remarkable similarity to that of a mutation in human timeless that underlies FASPS, our results may provide new insights into the molecular underpinnings of human FASPS.

neuroscience↗

bioRxiv2019

The HLA Ligand Atlas. A resource of natural HLA ligands presented on benign tissues.

The human leukocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Here, we describe the HLA Ligand Atlas, an extensive collection of mostly matched HLA-I and -II ligandomes from 225 benign samples (29 tissues, 21 subjects). The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 89,853 HLA-I- and 140,861 HLA-II ligands. We observe that the immunopeptidomes differ considerably between tissues and individuals on both source protein and HLA-ligand level. 1,407 HLA-I ligands stem from non-canonical genomic regions. We highlight the importance of comparatively analyzing both benign and malignant tissues to inform tumor association, based on a case study in three glioblastoma patients. The resource provides insights into applied and basic immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy, and autoimmunity. It is publicly available at www.hla-ligand-atlas.org.

immunology↗

bioRxiv2019

Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate

Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila lacks a gene for carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of Drosophila carboxypeptidase D (dCPD) in global neuropeptide processing and selected peptide-regulated behaviours. We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD-encoding gene silver in the larva causes lethality, and leads to deficits in adult starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced survival rate and activity levels. A phylogenomic analysis suggests that loss of CPE is not a common insect feature, but specifically occured in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing in Drosophila, and is required for proper peptide-regulated behaviour. dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. Our results raise the question why Drosophila and other Diptera and Hymenoptera -unlike other insects-have obviously lost the gene for CPE but kept a gene encoding CPD.

neuroscience↗