Biology subjects

Eckhardt, M.

Publications and source records attributed to Eckhardt, M..

11 recordsLinked to original sources

bioRxiv2025

Paracrine Signals from HIV-1 Infected Immune Cells Reprogram Cervical Cancer Pathways

Persistent infection with human papillomavirus (HPV) is the primary cause of cervical cancer worldwide. Notably, women co-infected with HPV and human immunodeficiency virus type 1 (HIV-1) have a six-fold higher lifetime risk of developing cervical cancer compared to those without HIV, even when adhering to antiretroviral therapy (ART) and achieving T-cell reconstitution. While chronic HIV-1 infection is known to cause inflammation, how paracrine signals from immune cells alter signaling in cervical cells remain poorly understood. To address this, we conducted global transcriptomics analysis on cervical swabs from Kenyan women with HPV, stratified by HIV-1 and cancer status. Strikingly, women with HIV-1 showed cancer-like gene expression patterns in non-cancerous cervical epithelial cells. Complementary global mass spectrometry (MS) proteomics of cervical cells exposed to the secretome of HIV-1 infected primary CD4+ T-cells revealed altered expression of proteins in MAPK, PI3K-AKT, and {beta}-catenin signaling pathways. Integrative network analyses of transcriptomic and proteomic datasets revealed that HIV-1 altered gene expression in key pathways known to drive cervical cancer, including genes commonly mutated in HIV-1-naive disease. Notably, IRS-1, a key PI3K-AKT pathway activator, was found to be consistently upregulated in both participant samples and cell culture models, as were interferon-stimulated genes. Phosphoproteomics MS analysis confirmed PI3K-AKT pathway activation in cervical cells exposed to conditioned media from HIV-1-infected T-cells. Together, our findings uncover how HIV-1 reshapes cervical cell signaling via paracrine mechanisms and highlights the PI3K pathway as a potential therapeutic target in HIV-associated cervical cancer.

cancer biology↗

bioRxiv2025

Impaired autophagic flux in skeletal muscle of plectin-related epidermolysis bullosa simplex with muscular dystrophy

BackgroundPlectin, a multi-functional cytolinker and intermediate filament (IF) stabilizing protein, is essential for muscle fiber integrity and function. Mutations in the human plectin gene (PLEC) cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). The disorganization and aggregation of desmin IFs in conjunction with degenerative changes of the myofibrillar apparatus are key features in the skeletal muscle pathology of EBS-MD. We performed a comprehensive analysis addressing protein homeostasis in this rare protein aggregation disease by using human EBS-MD tissue, plectin knock-out mice, and plectin-deficient cells. MethodsProtein degradation pathways were analyzed in muscles from EBS-MD patients, muscle-specific conditional plectin knockout (MCK-Cre/cKO) mice, as well as immortalized plectin-deficient (Plec-/-) myoblasts by electron and immunofluorescence microscopy. To obtain a comprehensive picture of autophagic processes, we evaluated the transcriptional regulation and expression levels of autophagic markers in plectin-deficient muscles and myoblasts (RNA-Seq, qRT-PCR, immunoblotting). Autophagic turnover was dynamically assessed by measuring baseline autophagy as well as specific inhibition and activation in mCherry-EGFP-LC3B-expressing Plec+/+and Plec-/- myoblasts, and by monitoring primary wild-type (WT) and plectin-deficient (P0) myoblasts using organelle-specific dyes. Analyses of chloroquine (CQ)-treated MCK-Cre/cKO mice corroborated that loss of plectin coincides with impaired autophagic clearance. ResultsOur study identified massive accumulation of degradative vacuoles as well as LC3 and SQSTM1-positive patches in EBS-MD patient and MCK-Cre/cKO mouse muscles and Plec-/- myoblasts. While the transcriptional regulation of autophagy-related proteins remained largely unaltered, protein levels of downstream targets of the autophagosomal degradation machinery were elevated in MCK-Cre/cKO muscle lysates (e.g. LAMP2, BAG3, and SQSTM1 to [~]160, [~]150, and [~]140% of control samples, respectively; P<0.05). Autophagosome turnover was compromised in mCherry-EGFP-LC3B-expressing Plec-/-myoblasts compared to Plec+/+ cells ([~]40% reduction in median red:green ratio, reduced puncta number, smaller puncta; P<0.01). By labelling autophagic compartments with CYTO-ID dye or lysosomes with LYSO-ID, we found reduced signal intensities in P0 cells (P<0.001). Treatment of primary myoblasts with CQ led to drastic swelling of autophagic vacuoles in WT myoblasts, while the swelling in P0 cells was moderate, establishing a defect in their autophagic clearance. Finally, CQ-treatment of MCK-Cre/cKO mice reassured in vivo the concept that autophagic flux is impaired in plectin-deficient muscles. ConclusionsOur work demonstrates that the characteristic protein aggregation pathology in EBS-MD is linked to an impaired autophagic flux. The obtained results open a new perspective on the understanding of the protein aggregation pathology in plectin-related disorders and provides a basis for further pharmacological intervention.

cell biology↗

bioRxiv2023

Deep MALDI-MS Spatial Omics guided by Quantum Cascade Laser Mid-infrared Imaging Microscopy

In spatial omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry (MS)-based spatial omics must compromise between data acquisition speed and biochemical profiling depth, thus often leading to only "putative" molecular identifications. Here, we introduce fast quantum cascade laser mid-infrared imaging microscopy to guide MS imaging to confined tissue areas of high interest, e.g., multicellular spheroid cores or kidney glomeruli, for spatial lipidomics profiling at maximized analytical depth utilizing magnetic resonance-MS imaging at >106 resolution or prm-PASEF-MS2 fragmentation imaging. Instigating selective sulfatide accumulation in arylsulfatase A-deficient mice as ground truth concept, we demonstrate that deep QCL-infrared-guided on-tissue spatial omics unequivocally identifies 120 sulfatides. This approach enables identifications of odd-chain sulfatides and studies of structure-ion mobility-relationships that provide chemical rationales for improvements to current ion mobility prediction algorithms. Workflows and data processing tools are provided as community resources.

biochemistry↗

bioRxiv2023

A foundational atlas of autism protein interactions reveals molecular convergence

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

systems biology↗

bioRxiv2023

An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping

Proximity labeling (PL) through biotinylation coupled with mass spectrometry (MS) has emerged as a powerful technique for capturing spatial proteomes within living cells. Large-scale sample processing for proximity proteomics requires a workflow that minimizes hands-on time while enhancing quantitative reproducibility. Here, we present a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. By combining this pipeline with an optimized quantitative MS acquisition method based on data-independent acquisition (DIA), we not only significantly increased sample throughput but also improved the reproducibility of protein identification and quantification. We applied this pipeline to delineate subcellular proteomes across various cellular compartments, including endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, employing 5HT2A serotonin receptor as a model, we investigated temporal changes of proximal interaction networks induced by the receptors activation with serotonin. Finally, to demonstrate the applicability of our PL pipeline across multiple experimental conditions, we further modified the PL pipeline for reduced sample input amounts to accommodate CRISPR-based gene knockout, and assessed the dynamics of the 5HT2A network in response to the perturbation of selected proximal interactors. Importantly, the presented PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.

systems biology↗

bioRxiv2023

Carnosine synthase deficiency aggravates neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, the presence of CARNS1 and endogenous HCDs does not protect against cuprizone-induced demyelination, and is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur. Exogenously administered carnosine is not effective in blunting demyelination or accelerating remyelination. In conclusion, we show that CARNS1 is diminished in demyelinated MS lesions, which may have detrimental effects on disease progression through weakening the endogenous protection against neuroinflammation.

neuroscience↗

bioRxiv2023

HBV rewires liver cancer signaling by altering PP2A complexes

Infection by hepatitis B virus (HBV) increases risk for liver cancer by inducing inflammation, cellular stress and cell death. To elucidate the molecular pathways by which HBV promotes cancer development and progression, we used affinity purification mass spectrometry to comprehensively map a network of 145 physical interactions between HBV and human host proteins in hepatocellular carcinoma (HCC). We find that viral proteins target host factors that are preferentially mutated in non-HBV-associated HCC, implicating cancer pathways whose interaction with HBV plays a role in HCC. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we show that HBx remodels the PP2A phosphatase complex, altering its effect on tumor signaling. HBx excludes striatin-family regulatory subunits from PP2A, causing Hippo kinase activation and unmasking a requirement for mTOR complex 2 to maintain expression of the YAP oncoprotein in HCC. Thus, HBV rewires HCC to expose potentially targetable signaling dependencies. SignificancePrecision medicine has revolutionized cancer treatment but remains elusive for HCC. We used proteomics to define HBV/host interactions and integrated them with HCC mutations. The results implicate modifiers of HCC behavior via remodeling of host complexes and illuminate new biological mechanisms in advanced disease for therapeutic investigation.

cancer biology↗

bioRxiv2022

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic--Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans. One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

systems biology↗

bioRxiv2022

Structure-function analysis of enterovirus protease 2A in complex with its essential host factor SETD3

Enteroviruses cause a number of medically relevant and widespread human diseases with no approved antiviral therapies currently available. Host-directed therapies present an enticing option for this diverse genus of viruses. We have previously identified the actin histidine methyltransferase SETD3 as a critical host factor physically interacting with the viral protease 2A. Here, we report the 3.5 [A] cryo-EM structure of SETD3 interacting with coxsackievirus B3 2A at two distinct interfaces, including the substrate-binding surface within the SET domain. Structure-function analysis revealed that mutations of key residues in the SET domain resulted in severely reduced binding to 2A and complete protection from enteroviral infection. Our findings provide insight into the molecular basis of the SETD3-2A interaction and a framework for the rational design of host-directed therapeutics against enteroviruses.

biophysics↗

bioRxiv2022

Quantitative proteomic analysis reveals apoE4-dependent phosphorylation of the actin-regulating protein VASP

Apolipoprotein (apo) E4 is the major genetic risk factor for Alzheimers Disease (AD). While neurons generally produce a minority of the apoE in the central nervous system, neuronal expression of apoE increases dramatically in response to stress and is sufficient to drive pathology. Currently, the molecular mechanisms of how apoE4 expression may regulate pathology are not fully understood. Here we expand upon our previous studies measuring the impact of apoE4 on protein abundance to include the analysis of protein phosphorylation and ubiquitylation signaling in isogenic Neuro-2a cells expressing apoE3 or apoE4. ApoE4 expression resulted in a dramatic increase in VASP S235 phosphorylation in a PKA-dependent manner. This phosphorylation disrupted VASP interactions with numerous actin cytoskeletal and microtubular proteins. Reduction of VASP S235 phosphorylation via PKA inhibition resulted in a significant increase in filopodia formation and neurite outgrowth in apoE4-expressing cells, exceeding levels observed in apoE3-expressing cells. Our results highlight the pronounced and diverse impact of apoE4 on multiple modes of protein regulation and identify protein targets to restore apoE4-related cytoskeletal defects.

neuroscience↗

bioRxiv2020

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

systems biology↗