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bioRxiv2026

Mapping the Mutational Landscape for Streptokinase Binding to Plasminogen

Group A Streptococcus (GAS) expresses streptokinase (SK), a critical virulence factor that non-enzymatically activates the hosts plasminogen (PLG), to an active form, PLGSK, resulting in the degradation of fibrin clots and subsequent bacterial dissemination. PLGSK formation contrasts with the physiologic activation of PLG to the serine protease plasmin via a proteolytic mechanism. As a potent thrombolytic, SK has been used as a therapeutic to treat heart attacks and strokes. GAS SK is highly specific for human PLG, and sequence variation between SK from different GAS strains has been linked to differences in PLG binding and disease severity. We now report the application of deep mutational scanning (DMS) to map the effects of [~]71% of single amino acid substitutions within SK from Group C Streptococcus, which shares high sequence homology with GAS on its ability to bind human PLG. We first demonstrate that SK expressed as a fusion protein to the p3 coat protein of M13 filamentous phage retains its capacity to bind human PLG. Our subsequent DMS analysis using this phage system identifies regions of SK in which amino acid substitutions are likely to increase or decrease its affinity for PLG. Our findings suggest a complex protein-protein interaction in which long-range protein dynamics influence the conformational activation of PLG to PLGSK. These data lay the foundation for linking SK variation between GAS strains to differences in virulence, mapping the determinants of GAS SKs human specificity, and potentially contributing to the development of improved therapeutics for heart attack and stroke.

microbiology↗

bioRxiv2026

Endothelial Cell Secretome Alterations Induced by Inflammatory Stress

Endothelial cells detect pathogens through pattern recognition receptors, such as Toll-like receptor 4 (TLR4), which triggers the synthesis and secretion of molecules that initiate the innate immune response. Proteins bearing signal peptides are secreted through the classical endoplasmic reticulum (ER)-Golgi-dependent route, whereas select signal-peptide-lacking cytoplasmic proteins are secreted via less well-characterized ER-Golgi-independent mechanisms, collectively termed unconventional cytoplasmic protein secretion (UCPS). To systematically characterize the secretome of human umbilical vein-derived endothelial cells (HUVECs) and delineate the contribution of UCPS, we performed deep quantitative proteomics on HUVEC cell lysates and conditioned medium before and after TLR4 stimulation with lipopolysaccharide (LPS). Of 5205 proteins detected in either fraction, 381 were enriched in the conditioned medium and therefore classified as secreted. Of these, 333 proteins (87.4%) were secreted via the conventional pathway, and 48 (12.6%) were secreted via UCPS, 43 of which were not previously associated with this process. Predicted functions of UCPS-secreted proteins include redox regulation, proteostasis, cytoskeletal remodeling, and innate immune signaling. We confirmed that -globin (HBA1), which functions as a redox sensor and regulator of nitric oxide in endothelial cells, is secreted constitutively by UCPS and at higher levels following inflammatory activation. Notably, UCPS cargo identity showed poor concordance with current computational predictors, underscoring the need for empirical datasets. Overall, our findings suggest that the HUVEC secretome includes both conventionally and unconventionally secreted proteins that regulate coagulation, angiogenesis, and immune function. Our findings establish a high-quality secretome dataset for HUVECs, providing a novel resource for future efforts to define the molecular determinants governing UCPS cargo selection and trafficking related to endothelial cell function.

cell biology↗