Biology subjects

Samiotaki, M.

Publications and source records attributed to Samiotaki, M..

18 recordsLinked to original sources

bioRxiv2026

Neurofibromin Regulates Sleep Consolidation via Cholinergic Neurons in a Drosophila Model of Neurofibromatosis 1

Neurofibromatosis type 1 (NF1) patients frequently present with fragmented, poor-quality sleep, but the neural circuits and molecular mechanisms underlying this deficit remain undefined. Using Drosophila, we show that loss of the NF1 ortholog dNf1 recapitulates the human phenotype: null mutants exhibit markedly reduced total sleep with blunted nighttime consolidation, reflected in fewer and shorter sleep bouts and elevated inter-bout activity, a recessive phenotype reproduced by independent null alleles and by pan-neuronal RNAi knockdown. Despite the broad expression of dNf1 across the adult CNS, including the mushroom bodies, ellipsoid body, dorsal fan-shaped body, and GABAergic neurons, cell-type-restricted knockdown showed the protein is dispensable for sleep regulation in each of these populations. Instead, dNf1 loss specifically in cholinergic neurons, the most abundant neuronal class in the fly brain, was sufficient to reproduce the deficient, fragmented sleep of null mutants, and restoring wild-type dNf1 to cholinergic neurons of null animals was both necessary and sufficient to rescue sleep. Mechanistically, cholinergic expression of the GAP-related domain (GRD) alone, which normalizes elevated Ras/MAPK signaling, fully restored sleep in nulls, whereas a catalytically dead GRD variant did not, establishing Ras/MAPK dysregulation in cholinergic neurons as the proximate cause. Two disease-associated missense mutations, dNf1C1045Y and dNf1R1861C, produced distinct, dose-graded sleep phenotypes. dNf1C1045Y showed severe deficits resembling the null and was resistant to pan-neuronal, but responded to cholinergic neuron-restricted rescue, consistent with a dominant-negative effect on dNf1 dimerization. In contrast, dNf1R1861C presented a milder phenotype that was largely reversible by wild-type dNf1 but only partially by the GRD domain, implicating an additional, GRD-independent mechanism. Collectively, these findings define cholinergic neurons as the key circuit through which Neurofibromin regulates sleep consolidation via Ras/MAPK signaling and reveal mutation-specific mechanisms that mirror the phenotypic heterogeneity seen in NF1 patients, with implications for genotype-informed understanding of sleep disturbance in this disorder.

neuroscience↗

bioRxiv2026

The loss of Tau in the adult brain triggers neuroplastic, epigenetic and behavioral deficits

INTRODUCTIONAccumulation of pathological Tau precipitates neuronal malfunction in Alzheimers disease (AD). However, the impact of loss of normal Tau function in the adult brain, independently of Tau aggregates, remains unclarified. METHODSWe used a mouse model with conditional mapt knocking-down in forebrain of 5-7 months old animals (cTau-KO), a virus-driven selective Tau knockdown in wild-types (WT) and Tau re-expression approaches, accompanied by neurostructural, epigenetic, proteomic, electrophysiological, neurochemical and behavioral analyses. RESULTSLoss of Tau in the adult brain of cTau-KOs triggers neuronal atrophy and malfunction, epigenetic as well cognitive and mood deficits. Importantly, these perturbations were confirmed by selective Tau loss in WT adult brain and reverted by Tau re-expression or pharmacologically-induced epigenetic correction in cTau-KOs. DISCUSSIONOur findings highlight the contribution of loss of normal Tau function in the adult brain malfunction that could be relevant in diverse brain pathologies beyond AD, associated to Tau and its dysfunction.

neuroscience↗

bioRxiv2025

Mena-dependent local translation of PI3K-p85 coordinates the axonal regenerative program upon injury

Local translation (LT) is one of the first processes activated after nerve injury and is critically coupled with the intrinsic ability of axons to grow and regenerate. However, regulation of LT in adult axons remains highly unexplored. Here we identify the actin regulator Mena, as a key mediator of adult axon regeneration, via its control of local protein synthesis. We show that Mena directly associates with PI3K-p85 mRNA and regulates its LT in injured sciatic nerve axons, thereby activating the downstream AKT/mTOR pathway. Genetic ablation of Mena disrupts this intrinsic response, resulting in impaired axonal LT and diminished axon regeneration in vivo. Our findings identify a Mena-dependent mechanism that remodels the local axonal proteome and reveal a critical link between mRNA regulation and metabolic signaling during axon regeneration.

neuroscience↗

bioRxiv2025

RpS12-mediated induction of the Xrp1short isoform links ribosomal protein mutations to cell competition

Cell competition, a universal yet enigmatic phenomenon, eliminates less-fit cells via interactions with their neighbors. It was originally described in Drosophila mosaics, where heterozygous ribosomal protein (Rp+/-) mutant cells are eliminated by wild-type neighbors. The transcription factor Xrp1 mediates most of the Rp+/- -associated phenotypes, including reduced competitiveness and translation. Although RpS12 is required for Xrp1 induction in Rp+/- cells, the mechanism remained unresolved. We demonstrate that RpS12, via alternative splicing, induces the Xrp1 short (Xrp1short) isoform expression in Rp+/- cells, which is both necessary and sufficient for their elimination. Strikingly, RpS12 overexpression in wild-type cells is sufficient to induce Xrp1short expression and confer a "loser" phenotype. While Xrp1long isoform is not required in Rp+/- cells, expression of either Xrp1 isoform is sufficient to promote the loser status in wild-type cells. We further identify Syncrip, an RNA-binding protein reduced in Rp+/- cells, as a critical Xrp1 suppressor; its depletion in wild-type cells activates Xrp1-dependent competition. Our findings establish RpS12s specialized function in Xrp1short promotion, not proteotoxic stress, as the primary driver in Rp+/- cells, providing new perspectives that challenge and refine prevailing models. Our work contributes in long-standing questions about ribosomal protein-linked fitness surveillance and provides insights into ribosomopathy pathologies.

developmental biology↗

bioRxiv2025

ERAP1 allotypes 2 and 10 differentially regulate the immunopeptidome of melanocytes

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a polymorphic enzyme that shapes the peptide repertoire presented by MHC class I molecules and can regulate adaptive immune responses in cancer and autoimmunity. Common missense polymorphisms in ERAP1 modulate its activity and are found in specific allotypes in humans. ERAP1 allotypes are linked to predisposition to HLA-associated inflammatory diseases. ERAP1 allotypic variation has been correlated with the development of psoriasis and Bechets disease, through the generation of specific CD8+ T cell populations targeting disease-specific HLAs. Given the established broad effects of ERAP1 activity on the cellular immunopeptidome, we hypothesized that ERAP1 allotypic variation may lead to broad immunopeptidome shifts that underlie antigenic responses. To test this hypothesis, we generated two A375 melanoma cell lines, each one expressing one of the most common, disease-associated, ERAP1 allotypes, namely allotypes 2 or 10. Comparison of the immunopeptidome of these two cell lines showed only minor differences in peptide sequences presented but extensive changes in abundance that included alterations in length distribution, binding affinity and sequence motifs. Our results suggest that enzymatic differences between ERAP1 allotypes are reflected primarily on the quantitative composition of the cellular immunopeptidome. These quantitative changes may constitute a mechanism that underlies ERAP1-allotypic associations with HLA-associated autoimmunity and variable anti-tumor responses.

immunology↗

bioRxiv2025

Exercise induces anti-inflammatory reprogramming in macrophages via Hsp60

Physical activity exerts systemic anti-inflammatory effects and reduces the risk for multiple non-communicable diseases, with 7.2% of all-cause deaths globally being attributed to physical inactivity. However, the cellular and molecular components of the exercise-induced anti-inflammatory effects remain only partly understood. Herein we show that moderate-intensity exercise promotes anti-inflammatory reprograming of macrophages orchestrated by the skeletal muscle cells secretome. Primary bone marrow-derived macrophages (BMDMs) exposed to the secretome of mechanically-loaded myotubes (exercise-conditioned medium, exCM) acquire an anti-inflammatory transcriptional profile and increased reliance on oxidative phosphorylation, as shown by Seahorse real-time cell metabolic analysis, compatible with an M2-like phenotypic switch. Using an unbiased proteomic analysis of the exCM we identify the chaperonin Hsp60 as a key mediator of the anti-inflammatory effects of exercise. Hsp60 expression increases in mechanically loaded myotubes in vitro, in the quadriceps muscle and serum of mice following an 8-week program of moderate-intensity aerobic exercise, as well as in human muscle after resistance training. Importantly, treatment of BMDMs with Hsp60 in vitro recapitulates the exCM-induced transcriptional reprograming, promoting an M2-like phenotype. Taken together, our data highlight Hsp60 as a novel component of the skeletal muscle cell-macrophage crosstalk, providing mechanistic insights into the anti-inflammatory effects of exercise.

physiology↗

bioRxiv2024

Proteostasis dysregulation in p.A53T-α-Synuclein iPSC-derived astrocytes exacerbates neurodegeneration in a Parkinson's disease model with Lewy-like pathology

Alpha-Synuclein (Syn) plays a central role in Parkinsons disease (PD) and the p.A53T mutation causes an early-onset familial form of PD with severe manifestations. The pathological effects of the p.A53T-Syn mutation have been extensively investigated in neurons, yet the consequences on astrocytes and astrocytic contribution to PD pathology are understudied. Here, we differentiated induced pluripotent stem cells from PD patients carrying the p.A53T-Syn mutation to astrocytes, which uncovered cell-intrinsic phenotypes, including calcium dyshomeostasis and accumulation of protein aggregates. Proteomic profiling and functional analyses revealed perturbed protein catabolic processes, involving the proteasome and autophagy, associated with lysosomal malfunction. Dopamine neurons co-cultured with p.A53T-Syn astrocytes displayed exacerbated neurodegeneration with hallmark Lewy-like pathologies, reversed by control astrocytes at least due to their ability to resolve neuronal Syn aggregates by endocytic clearance. Our findings underscore a critical impact of p.A53T-Syn on astrocytic protein quality control mechanisms, positioning astrocytes as important contributors to PD neuropathology. HighlightsO_LIiPSC-derived astrocytes from PD patients with the p.A53T-Syn mutation display cell-autonomous pathological phenotypes and toxic Syn accumulation C_LIO_LIProteome and functional analyses reveal failure of major proteostasis mechanisms in p.A53T-Syn astrocytes, largely related to lysosomal malfunction C_LIO_LIInherent Lewy-like pathological features are identified in p.A53T-Syn astrocyte-neuron co-cultures C_LIO_LIp.A53T-Syn astrocytes induce PD-relevant neuropathology to healthy neurons C_LIO_LIControl but not p.A53T-Syn astrocytes alleviate neuropathology in co-cultured neurons C_LI

neuroscience↗

bioRxiv2024

ERAP1 activity modulates the immunopeptidome but also affects the proteome, metabolism and stress responses in cancer cells

Endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) metabolizes peptides inside the ER and shapes the peptide repertoire available for binding to Major Histocompatibility Complex Class I molecules (MHC-I). Moreover, it may have additional effects on cellular homeostasis, which have not been explored. To address these questions, we used both genetic silencing of ERAP1 expression as well as treatment with a selective allosteric ERAP1 inhibitor to probe changes in the immunopeptidome and proteome of the A375 melanoma cancer cell line. We observed significant immunopeptidome shifts with both methods of functional ERAP1 disruption, which were distinct for each method. Both methods of inhibition led to an enhancement, albeit slight, in tumor cell killing by stimulated human PBMCs and in significant proteomic alterations in pathways related to metabolism and cellular stress. Similar proteomic changes were also observed in the leukemia cell line THP-1. Biochemical analyses suggested that ERAP1 inhibition affected sensitivity to ER stress, reactive oxygen species production and mitochondrial metabolism. Although the proteomics shifts were significant, their potential in shaping immunopeptidome shifts was limited since only 15.8% of differentially presented peptides belonged to proteins with altered expression and only 5.0% of proteins with altered expression were represented in the immunopeptidome shifts. Taken together, our findings suggest that modulation of ERAP1 activity can generate unique immunopeptidomes, mainly due to altered peptide processing in the ER, but also induce changes in the cellular proteome and metabolic state which may have further effects on tumor cells.

biochemistry↗

bioRxiv2024

GABA/Glutamate neuron differentiation imbalance and increased AKT/mTOR signalling in CNTNAP2-/-cerebral organoids.

We developed a human cerebral organoid model derived from induced pluripotent stem cells (iPSCs) with targeted genome editing to abolish protein expression of the Contactin Associated Protein-like 2 (CNTNAP2) autism spectrum disorder (ASD) risk gene, mimicking loss-of-function mutations seen in patients. CNTNAP2-/- cerebral organoids displayed accelerated cell cycle, ventricular zone disorganisation and increased cortical folding. Proteomic analysis revealed disruptions in Glutamatergic/GABAergic synaptic pathways and neurodevelopment, highlighting increased protein expression of corticogenesis and neurodevelopment-related genes such as Forkhead box protein G1 (FOXG1) and Paired box 6 (PAX6). Transcriptomic analysis revealed differentially expressed genes (DEG) belonging to inhibitory neuron-related gene networks. Interestingly, there was a weak correlation between the transcriptomic and proteomic data, suggesting nuanced translational control mechanisms. Along these lines we found upregulated Protein Kinase B (Akt)/mechanistic target of rapamycin (mTOR) signalling in CNTNAP2-/- organoids. Spatial transcriptomics analysis of CNTNAP2-/- ventricular-like zones demonstrated pervasive changes in gene expression, particularly in PAX6- cells, implicating upregulation of cell cycle regulation pathways, synaptic and Glutamatergic/GABAergic pathways. We noted a significant overlap of all D30 cerebral organoids omics datasets with an idiopathic ASD (macrocephaly) iPSC-derived telencephalic organoids DEG dataset, where FOXG1 was upregulated. Moreover, we detected increased Glutamate decarboxylase 1 (GAD1) and decreased T-Box Brain Transcription Factor 1 (TBR1) expressing cells, suggesting altered GABAergic/Glutamatergic neuron development. These findings potentially highlight a shared mechanism in the early cortical development of various forms of ASD, further elucidate the role of CNTNAP2 in ASD pathophysiology and cortical development and pave the way for targeted therapies using cerebral organoids as preclinical models.

neuroscience↗

bioRxiv2023

An Immunological Synapse Formation Between T Regulatory Cells and Cancer-Associated Fibroblasts Promotes Tumor Development

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumor microenvironment, serving diverse functions in tumor progression, invasion, matrix remodeling and resistance to therapy; yet, the precise mechanisms via which CAFs imprint on the anti-tumor immunity remain poorly understood. Extensive molecular characterization revealed an increased heterogeneity in the CAF compartment and proposed an interaction between CAFs and tumor-infiltrating immune cells, which may shape tumor immune evasion. Herein, we describe a synapse formation between -SMA+ CAFs and regulatory T cells (Tregs) in the TME. Foxp3+ Tregs were localized close to -SMA+ CAFs in diverse types of tumor models as well as biopsies from melanoma and colorectal cancer patients. Notably, phenotypically tolerogenic -SMA+ CAFs demonstrated the ability to phagocytose and process tumor antigens, instructing Treg movement arrest, activation and proliferation, in an antigen-specific manner. Of interest, -SMA+ CAFs were characterized by the presence of double-membrane structures, resembling autophagosomes, in their cytoplasm, while analysis of single-cell transcriptomic data pointed autophagy and antigen processing/presentation pathways to be enriched in -SMA-expressing CAF clusters. In a mechanistic view, conditional knockout of the autophagy pathway in -SMA+ CAFs promoted an inflammatory re-programming of CAFs, reduced Treg infiltration, attenuated tumor development, and potentiated the efficacy of immune checkpoint inhibitor immunotherapy. Overall, our findings reveal an immunosuppressive mechanism operating in the TME, which entails the formation of synapses between -SMA+ CAFs and Tregs in an autophagy-dependent fashion and raises the potential for the development of CAF-targeted therapies in cancer. One Sentence Summary-SMA+ Cancer Associated Fibroblasts process and present tumor antigens via autophagy to form immunological synapses with Foxp3+ T regulatory cells in the tumor microenvironment, promoting tumor development.

cancer biology↗

bioRxiv2023

Chronic optogenetic activation of hippocampal pyramidal neurons replicates the proteome footprint of Alzheimer's disease-like pathology

Neuronal overexcitability can elicit synaptic changes, leading to neuronal hyperactivity and abnormal neural circuit processing. Such network disruption impairs neuronal function and survival, initiating neurodegeneration and Alzheimers disease (AD). Yet, the sequence of synaptic changes resulting from excessive neuronal activity remains elusive. We employed optogenetics to induce sustained neuronal hyperactivity in the hippocampi of wild-type and AD-like 5xFAD mice. Surprisingly, after a month of daily optogenetic stimulation, the proteomic profiles of photoactivated wild-type and 5xFAD mice exhibited remarkable similarity. Proteins involved in translation, protein transport, autophagy, and notably in the AD pathology were upregulated in wild-type mice. Conversely, both glutamatergic and GABAergic synaptic proteins were downregulated. These hippocampal proteomic and signaling alterations in wild-type mice resulted in spatial memory loss and augmented A{beta}42 secretion. Collectively, these findings indicate that sustained neuronal hyperactivity alone replicates proteome changes seen in AD-linked mutant mice. Therefore, prolonged neuronal hyperactivity may contribute to synaptic transmission disruption, memory deficits and the neurodegenerative process associated with AD.

neuroscience↗

bioRxiv2023

The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking

Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst. The Golgi-dependent secretory pathway is thought to be responsible for the trafficking of all major membrane proteins. However, our recent findings in Aspergillus nidulans showed that several plasma membrane cargoes, such as transporters and receptors, follow a sorting route that seems to bypass Golgi functioning. To gain insight on membrane trafficking and specifically Golgi-bypass, here we used proximity dependent biotinylation (PDB) coupled with data-independent acquisition mass spectrometry (DIA-MS) for identifying transient interactors of the UapA transporter. Our assays, which included proteomes of wild-type and mutant strains affecting ER-exit or endocytosis, identified both expected and novel interactions that might be physiologically relevant to UapA trafficking. Among those, we validated, using reverse genetics and fluorescence microscopy, that COPI coatomer is essential for ER-exit and anterograde trafficking of UapA and other membrane cargoes. We also showed that ArfAArf1 GTPase activating protein (GAP) Glo3 contributes to UapA trafficking at increased temperature. This is the first report addressing the identification of transient interactions during membrane cargo biogenesis using PDB and proteomics coupled with fungal genetics. Our work provides a basis for dissecting dynamic membrane cargo trafficking via PDB assays. HighlightsO_LIA PDB-proteomics system for identifying transient interactions in A.nidulans C_LIO_LIUapA interactomes in wild-type and specific mutants C_LIO_LIAnalysis and validation of UapA interacting proteins C_LIO_LICOPI and Arf1 are essential for cargo anterograde trafficking C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/550021v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@c56a44org.highwire.dtl.DTLVardef@c39be2org.highwire.dtl.DTLVardef@2f264forg.highwire.dtl.DTLVardef@17c4820_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2023

Ferroptosis-Protective Membrane Domains in Quiescence

Quiescence is a common cellular state, required for stem-cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that Flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging, defective quiescence maintenance, and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFA). FLPs are essential for the extra-mitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work is the first description of ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor for the long-term survival of quiescent cells.

cell biology↗

bioRxiv2022

A wide foodomics approach coupled with metagenomics elucidates the environmental signature of Protected Geographical Indication potatoes

The term "terroir" has been widely employed to link differential geographic phenotypes with sensorial signatures of agricultural food products, influenced by agricultural practices, soil type and climate. Nowadays, the Geographical Indications labeling has been developed to safeguard the quality of plant-derived food that is linked to a certain terroir and is generally considered as an indication of superior organoleptic properties and phytochemical profile. As the dynamics of agroecosystems are highly intricate, consisting of tangled networks of interactions between plants, microorganisms, and the surrounding environment, the recognition of the key molecular components of terroir fingerprinting remains a great challenge to protect both the origin and the safety of food commodities. Furthermore, the contribution of microbiome as a potential driver of the terroir signature has been underestimated until recently. Herein, we present a first comprehensive view of the multi-omic landscape related to transcriptome, proteome, epigenome, and metagenome of the popular Protected Geographical Indication potatoes of Naxos.

plant biology↗

bioRxiv2022

Communication between the nucleus and the mitochondria via NDUFS4 alternative splicing in gastric cancer cells

A constant communication between the nucleus and the mitochondria allows both organelles to ensure cellular homeostasis and adaptation to mitochondrial stress. Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving a large number of nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes are known, however alternative splicing (AS) regulation has not been implicated in this communication. Here, we show that IQGAP1, a scaffold protein that regulates AS of distinct subsets of genes in gastric cancer cells, participates in AS regulation that strongly affects mitochondrial respiration. Combined proteomic analyses and RNA-seq profiles of IQGAP1KO and parental cells show that IQGAP1KO alters a specific AS event of the mitochondrial respiratory chain complex I subunit NDUFS4 and downregulates a subset of complex I subunits. In IQGAP1KO cells, respiratory complex I intermediates accumulate resembling assembly deficiencies observed in patients with Leigh syndrome bearing NDUFS4 mutations. Mitochondrial complex I activity is significantly lower in KO compared to parental cells, while exogenous expression of IQGAP1 partially restores NDUFS4 AS pattern and expression and reverses mitochondrial defects of IQGAP1KO cells. Our work sheds light to a novel facet of IQGAP1 in mitochondrial quality control that involves fine-tuning of complex I activity through AS regulation.

molecular biology↗

bioRxiv2021

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 Spike Glycoprotein

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both SARS-CoV-2 infection severity and long-term immunity, either after recovery or vaccination. A hallmark of COVID-19 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides by 10 common allotypes of ER aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for MHC class I molecules, including known SARS-CoV-2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses to COVID-19.

biochemistry↗

bioRxiv2020

Generation of SARS-CoV-2 S1 spike glycoprotein putative antigenic epitopes in vitro by intracellular aminopeptidases

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2 and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.

biochemistry↗

bioRxiv2020

High Content Screening and Proteomic Analysis Identify the Kinase Inhibitor BX795 as a Potent Neuroprotective Compound in a Patient-Derived Model of Parkinson's Disease

Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)-based disease modeling represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinsons disease bearing the G209A (p.A53T) -synuclein (Syn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores disease-associated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the protective effects of BX795, comprising a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-Syn in neuronal cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have identified a promising small molecule with neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.

neuroscience↗