Biology subjects

Siemieniak, D.

Publications and source records attributed to Siemieniak, D..

7 recordsLinked to original sources

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↗

bioRxiv2025

Thermodynamics and selection of the plasminogen activator inhibitor-1 latency transition

Plasminogen activator inhibitor-1 (PAI-1), like other serine protease inhibitors (SERPINs), exists in a functionally active metastable conformation. Unlike other SERPINs, PAI-1 undergoes a relatively rapid rearrangement to a lower energy latent conformation. We employ deep mutational scanning (DMS) to simultaneously probe the effects of nearly all single amino acid substitutions (92%) in PAI-1 on its latency transition as well as its ability to inhibit its canonical protease target, urokinase-type plasminogen activator (uPA). The DMS results are interpreted in the context of variant effect predictors (EVE, AlphaMissense, and CPT) and protein stability predictors (EvoEF and FoldX), as well as the evolutionary conservation of the PAI-1 sequence space across extant vertebrate species. We demonstrate that while variant effect predictors can generally partition PAI-1 as functional or non-functional with respect to uPA inhibition, they perform poorly when attempting to discriminate the effects of PAI-1 variants on its latency transition. However, by employing protein stability predictors, we demonstrate that the PAI-1 latency transition is most likely driven by changes in the energy barrier to the latency transition. Finally, we show that PAI-1s ability to inhibit uPA, as well as its thermodynamic instability in its active conformation are both under purifying selection that limits the sequence space available to PAI-1. These findings suggest that DMS of collateral fitness effects, including PAI-1s latency transition, may be better interpreted in contexts other than variant effect predictors, including protein thermodynamics and evolution.

biochemistry↗

bioRxiv2024

Synergy of LPS and Shiga-toxin exposure on the RNA expression program of cultured human umbilical vein endothelial cells.

BackgroundHemolytic uremic syndrome (HUS) is a primary thrombotic microangiopathy that can cause acute renal failure, particularly in children, and is commonly triggered by Shiga toxin-producing Escherichia coli (STEC) infection. Although previous in vitro studies have shown that Shiga toxin (STX) can directly affect the endothelium, gram-negative bacteria such as E. coli also contain lipopolysaccharide (LPS) which is known to independently activate endothelial cells. Furthermore, studies in mice and baboons have shown a synergistic effect of LPS and STX in inducing HUS when administered simultaneously. ObjectiveDetermine how LPS, STX, or combined LPS/STX treatment affect the gene expression program of endothelial cells. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with STX, LPS or a combination of STX and LPS for either 4 or 24 hours, after which unbiased RNA sequencing analysis was performed. ResultsFour-hour treatment with LPS resulted in a greater number of differentially expressed genes as compared to treatment with STX. In contrast, 24-hour treatment with LPS or STX induced similar numbers of differentially expressed genes. Dual treatment with both toxins resulted in a synergistic effect, showing significant up-or downregulation of multiple transcripts that were not observed in either single treatment group, particularly at the 24-hour timepoint. ConclusionOur data provide a resource for assessing the role of endothelial cell gene expression in the pathogenesis of STEC infection and suggest that the responses of these cells to LPS, STX, or combined LPS/STX treatment are unique and temporally regulated.

physiology↗

bioRxiv2024

High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases

While members of large paralogous protein families share structural features, their functional niches often diverge significantly. Serine protease inhibitors (SERPINs), whose members typically function as covalent inhibitors of serine proteases, are one such family. Plasminogen activator inhibitor-1 (PAI-1) is a prototypic SERPIN, which canonically inhibits tissue-and urokinase-type plasminogen activators (tPA and uPA) to regulate fibrinolysis. PAI-1 has been shown to also inhibit other serine proteases, including coagulation factor XIIa (FXIIa) and transmembrane serine protease 2 (TMPRSS2). The structural determinants of PAI-1 inhibitory function toward these non-canonical protease targets, and the biological significance of these functions, are unknown. We applied deep mutational scanning (DMS) to assess the effects of [~]80% of all possible single amino acid substitutions in PAI-1 on its ability to inhibit three putative serine protease targets (uPA, FXIIa, and TMPRSS2). Selection with each target protease generated a unique PAI-1 mutational landscape, with the determinants of protease specificity distributed throughout PAI-1s primary sequence. Next, we conducted a comparative analysis of extant orthologous sequences, demonstrating that key residues modulating PAI-1 inhibition of uPA and FXIIa, but not TMPRSS2, are maintained by purifying selection. PAI-1s activity toward FXIIa may reflect how protease evolutionary relationships predict SERPIN functional divergence, which we support via a cophylogenetic analysis of all secreted SERPINs and their cognate serine proteases. This work provides insight into the functional diversification of SERPINs and lays the framework for extending these studies to other proteases and their regulators.

evolutionary biology↗

bioRxiv2022

Hepatic inactivation of murine Surf4 results in marked reduction in plasma cholesterol

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by {bsim}60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding {bsim}50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B. Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.

physiology↗

bioRxiv2019

The ER cargo receptor SURF4 facilitates efficient erythropoietin secretion

Erythropoietin (EPO), a glycoprotein produced by specialized peritubular fibroblasts in the kidney, is the master regulator of erythropoiesis. EPO is secreted into the plasma in response to tissue hypoxia and stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels. Using this cell line, we performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, consistent with a role for SURF4 as an ER cargo receptor of EPO, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO, ruling out an artifact of heterologous overexpression. This work suggests that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoeisis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

cell biology↗

bioRxiv2017

Whole exome sequencing of ENU-induced thrombosis modifier mutations in the mouse

Although the Factor V Leiden (FVL) gene variant is the most prevalent genetic risk factor for venous thrombosis, only 10% of FVL carriers will experience such an event in their lifetime. To identify potential FVL modifier genes contributing to this incomplete penetrance, we took advantage of a perinatal synthetic lethal thrombosis phenotype in mice homozygous for FVL (F5L/L) and haploinsufficient for tissue factor pathway inhibitor (Tfpi+/-) to perform a sensitized dominant ENU mutagenesis screen. Linkage analysis conducted in the 3 largest pedigrees generated from the surviving F5L/L Tfpi+/- mice ( rescues) using ENU-induced coding variants as genetic markers was unsuccessful in identifying major suppressor loci. Whole exome sequencing was applied to DNA from 107 rescue mice to identify candidate genes enriched for ENU mutations. A total of 3,481 potentially deleterious candidate ENU variants were identified in 2,984 genes. After correcting for gene size and multiple testing, Arl6ip5 was identified as the most enriched gene, though not reaching genome-wide significance. Evaluation of CRISPR/Cas9 induced loss of function in the top 6 genes failed to demonstrate a clear rescue phenotype. However, a maternally inherited (not ENU-induced) de novo mutation (Plcb4R335Q) exhibited significant co-segregation with the rescue phenotype (p=0.003) in the corresponding pedigree. Thrombosis suppression by heterozygous Plcb4 loss of function was confirmed through analysis of an independent, CRISPR/Cas9-induced Plcb4 mutation (p=0.01).\n\nAuthor summaryAbnormal blood clotting in veins (venous thrombosis) or arteries (arterial thrombosis) are major health problems, with venous thrombosis affecting approximately 1 in every thousand individuals annually in the United States. Susceptibility to venous thrombosis is governed by both genes and environment, with approximately 60% of the risk attributed to genetic influences. Though several genetic risk factors are known, >50% of genetic risk remains unexplained. Approximately 5% of people carry the most common known risk factor, Factor V Leiden. However, only 10% of these individuals will develop a blood clot in their lifetime. Mice carrying two copies of the Factor V Leiden mutation together with a mutation in a second gene called tissue factor pathway inhibitor develop fatal thrombosis shortly after birth. To identify genes that prevent this fatal thrombosis, we studied a large panel of mice carrying inactivating gene changes randomly distributed throughout the genome. We identified several genes as potential candidates to alter blood clotting balance in mice and humans with predisposition to thrombosis, and confirmed this protective function for DNA changes in one of these genes (Plcb4).

genetics↗