Biology subjects

Shamorkina, T. M.

Publications and source records attributed to Shamorkina, T. M..

6 recordsLinked to original sources

bioRxiv2026

Host cell remodeling via cyclin dependent kinases drives Ebola virus replication and transcription

Ebola virus (EBOV) causes re-emerging outbreaks of viral hemorrhagic fever with high case-fatality rates. As a negative-sense single stranded RNA virus, EBOV depends on its RNA-dependent RNA polymerase (L protein) to transcribe and replicate the viral genome. This takes place in cytoplasmic inclusion bodies that form following host cell remodeling involving yet unknown signaling pathways. Here, using mass spectrometry-based (phospho-)proteomics, we profiled global protein abundance and site-specific phosphorylation in HEK293T cells expressing an EBOV minigenome system. Our data reveal EBOV-induced rewiring of the host proteome and phosphorylation signaling landscape, including perturbations in cell cycle control, cytoskeletal organization, innate immune regulation, and DNA damage response. Kinase network analysis revealed that Hippo pathway kinases and especially cyclin dependent kinases like CDK2 are central drivers of EBOV replication and transcription. The functional necessity of these signaling pathways is demonstrated via inhibition of CDK family kinases with small molecule inhibitors, which halted EBOV minigenome replication and transcription when administered in the low micromolar range, demonstrating that these pathways represent putative antiviral targets.

molecular biology↗

bioRxiv2025

Deep Coverage and Extended Sequence Reads Obtained with a Single Archaeal Protease Expedite de novo Protein Sequencing by Mass Spectrometry

The ability to sequence proteins without reliance on a genomic template defines a critical frontier in modern proteomics. This approach, known as de novo protein sequencing, is essential for applications such as antibody sequencing, microbiome proteomics, and antigen discovery, which require accurate reconstruction of peptide and protein sequences. While trypsin remains the gold-standard protease in proteomics, its restricted cleavage specificity limits peptide diversity. This constraint is especially problematic in antibody sequencing, where the functionally critical regions often lack canonical tryptic sites. As a result, exclusively trypsin-based approaches yield sparse reads, leading to sequence gaps. Multi-protease and hybrid-fragmentation strategies can improve the sequence coverage, but they add complexity, compromise scalability and reproducibility. Here, we explore two HyperThermoacidic Archaeal (HTA)-proteases as single-enzyme solutions for de novo antibody sequencing. Each HTA-protease generated about five times more unique peptide reads than trypsin or chymotrypsin, providing high redundancy across all CDRs. Combined with EAciD fragmentation on a ZenoTOF 7600 system, this approach enabled complete, unambiguous antibody sequencing. De novo analysis using PEAKS/DeepNovo and Stitch showed up to fourfold higher alignment scores and reduced the sequence errors within the HTA-generated data. Additionally, the HTA-EAciD approach offers short digestion times, eliminates extensive cleanup, and enables analysis in a single LC-MS/MS run. This streamlined, single-protease approach delivers therefore performance comparable to multi-enzyme workflows, offering a scalable and efficient strategy for de novo protein sequencing across diverse applications.

biochemistry↗

bioRxiv2025

Cetacean coronavirus spikes highlight S glycoprotein structural plasticity

Coronaviruses (CoVs) exhibit a remarkable ability for spill-over infections into naive host populations. While much research has focused on the spike (S) glycoproteins of zoonotic alpha- and betacoronaviruses, the S proteins of gamma- and deltacoronaviruses, which predominantly infect avian hosts, remain poorly understood. Here, we present high-resolution cryo-EM structures of S proteins from two distinct gammacoronaviruses (75.7% sequence identity) that atypically infect marine mammals and belong to the Gammacoronavirus delphinapteri species. The cryo-EM reconstructions reveal that the spikes exhibit a unique quaternary architecture that distinguishes them from other coronaviruses. The S protein features a previously unidentified, tripodal quaternary assembly of the S1 subunit, in which S1B domains are presented in an upright position while their putative receptor binding sites are shielded by extended loops from the S1A domain of the same protomers. Additionally, the CeCoV spike proteins have evolved an additional and unique [~]200 residue N-terminal domain (S10). S10 lacks homology to known protein sequences but displays structural similarity to members of the cupin protein superfamily. This represents a remarkable case of coronaviral exaptation of a host protein integrated into the S glycoprotein. Moreover, glycoproteomic analyses reveal that CeCoV S proteins are extensively N-glycosylated (>100 N-glycans per trimer), with a notable abundance of high-mannose glycans on S10 and O-glycosylation sites within a mucin-like loop at the trimer apex, all contributing to a dense glycan shield and potentially masking immunogenic epitopes. These findings demonstrate the structural diversity and adaptability of CoV S proteins, including alternative quaternary assemblies, additional domains, and diverse glycosylation strategies, offering new insights into the evolutionary mechanisms that enable coronaviruses to expand their host range and establish infections in novel species.

microbiology↗

bioRxiv2024

Foamy microglia link oxylipins to disease progression in multiple sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by expanding demyelinating lesions, leading to severe and irreversible disability. The mechanisms driving lesion expansion, however, remain poorly understood. Here, using a multi-omics approach, we identified foamy microglia as primary contributors to the molecular profile of lesions and disease progression in secondary progressive MS. Lesions with foamy microglia are marked by the accumulation of cholesterol esters, bismonoacylglycerolphosphates (BMP), and oxylipins, along with high B-cell infiltration, increased levels of immunoglobulin G1, and elevated expression of Fc{gamma}- and complement receptors. Lesions with foamy GPNMB+-microglia display markers of enhanced phagocytosis, lipid metabolism, lysosomal dysfunction, and antigen presentation, but lack classical pro-inflammatory markers. Our data suggest that sustained phagocytosis of myelin overwhelms microglial endo-lysosomal capacity, leading to lipid droplet and oxylipin formation. This microglial phenotype may induce further recruitment of adaptive immune cells, axonal damage, drive lesion expansion and prevent remyelination. Monoacylglycerol lipase, involved in producing oxylipin precursors, was identified as a potential therapeutic target to disrupt this cycle and prevent chronic lesion expansion.

neuroscience↗

bioRxiv2024

Proteolytic activity of surface exposed HtrA determines its expression level and is needed to survive acidic conditions in Clostridioides difficile

To survive in the host, pathogenic bacteria need to be able to react to the unfavourable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.

microbiology↗

bioRxiv2020

Cryo-EM structure of coronavirus-HKU1 haemagglutinin esterase reveals architectural changes arising from prolonged circulation in humans

The human betacoronaviruses HKU1 and OC43 (subgenus Embecovirus) arose from separate zoonotic introductions, OC43 relatively recently and HKU1 apparently much longer ago. Embecovirus particles are studded with two types of surface projections called S (for spike) and HE (for haemagglutinin-esterase), with S mediating receptor-binding and membrane fusion and HE acting as a receptor-destroying enzyme. Together, they promote dynamic virion attachment to glycan-based receptors with 9-O-acetylated sialic acid as main constituent. We recently showed that adaptation of HKU1 and OC43 to replication in the human respiratory tract involved loss-of-function mutations in the lectin domain of HE. Here we present the cryo-EM structure of the [~]80 kDa, heavily glycosylated HKU1 HE at a global resolution of 3.4 [A]. Comparison to existing HE structures reveals a drastically truncated lectin domain, incompatible with sialic acid binding, but with the structure and function of the HE esterase domain left intact. Our cryo-EM structure, in combination with mass spectrometry analysis, also describes the extent of glycosylation on the now redundant lectin domain, which forms a putative glycan shield. The findings further our insight into the evolution and host adaptation of human embecoviruses and also demonstrate the utility of cryo-EM for studying small, heavily glycosylated proteins which are intractable to X-ray crystallography.

microbiology↗