Biology subjects

Meyers, W. M.

Publications and source records attributed to Meyers, W. M..

7 recordsLinked to original sources

bioRxiv2026

Single-Cell Analytics for Dose Response (SCADR) discriminates PTEN missense variants by lipid and protein phosphatase dysfunction

The proliferation of sequencing efforts has revealed a vast and expanding catalog of single nucleotide gene variants, many associated to, but with unclear roles in disease. Fully charactering variant impacts and linking specific protein dysfunctions to disease are challenging due to the multi-functional nature of many proteins and varying degree of variant effects on these functions. Lagging are sensitive approaches to empirically assess the impact of missense variant-induced single amino acid changes on a wide range of protein functions. To address these issues, we have developed an open-source computational analysis tool called SCADR (Single-Cell Analytics for Dose Response) for simultaneously measuring and comparing impacts of exogenously-expressed variants on multiple signaling pathways using multiplex phospho-antibody spectral flow cytometry in human cell lines. SCADR retains and correlates single-cell measures of signal protein activity states along with expression levels of exogenously-expressed variants, providing rich characterization of multiple protein functions, signaling protein interactions, and enhanced discrimination of variant impacts on different signaling pathways, highlighting each variants unique dysfunction profile. Here, we apply SCADR for analyses of the impact of 6 variants of the tumor-suppressor protein PTEN (P38H, C124S, G129E, Y138L, D268E, 4A) expressed in HEK293 cells on the phosphorylation states of the canonical and noncanonical downstream signaling proteins Akt, S6, CREB, ERK, and p38 detected with fluorophore-conjugated phospho-antibodies, along with an antibody detecting an N-terminal HA tag on PTEN variants allowing measures of dose-response effects of each variants expression on signaling cascades. Results identify variant-specific impacts on downstream signaling cascades.

genomics↗

bioRxiv2024

SARS-CoV-2 EndoU-ribonuclease regulates RNA recombination and impacts viral fitness.

Coronaviruses (CoVs) maintain large RNA genomes that frequently undergoes mutations and recombination, contributing to their evolution and emergence. In this study, we find that SARS-CoV-2 has greater RNA recombination frequency than other human CoVs. In addition, coronavirus RNA recombination primarily occurs at uridine (U)-enriched RNA sequences. Therefore, we next evaluated the role of SARS-CoV-2 NSP15, a viral endonuclease that targets uridines (EndoU), in RNA recombination and virus infection. Using a catalytically inactivated EndoU mutant (NSP15H234A), we observed attenuated viral replication in vitro and in vivo. However, the loss of EndoU activity also dysregulated inflammation resulting in similar disease in vivo despite reduced viral loads. Next-generation sequencing (NGS) demonstrated that loss of EndoU activity disrupts SARS-CoV-2 RNA recombination by reducing viral sub-genomic message but increasing recombination events that contribute to defective viral genomes (DVGs). Overall, the study demonstrates that NSP15 plays a critical role in regulating RNA recombination and SARS-CoV-2 pathogenesis.

microbiology↗

bioRxiv2024

Evolution of sex hormone-binding globulin gene expression in the primate testis

In lower mammals testicular sex hormone-binding globulin (SHBG), also known as androgen binding protein, is well known to be a product of the Sertoli cells. However in humans, testicular SHBG is a product of the germ cells, is expressed from an upstream promoter and contains an alternative first exon 1A. Examination of testicular SHBG transcripts from members across primate suborders revealed that transcripts containing exon 1A are unique to Hominoids and Old World Monkeys. In contrast testicular SHBG transcripts in gray mouse lemur contained the proximal exon 1, while no evidence for SHBG expression could be detected in marmoset monkey testes. In general, the exonic identity of primate testicular SHBG transcripts could be predicted based on the structure of their genes 5 regulatory region and we show that they change through the primate clade. This work provides insights into how molecular evolution of higher primate SHBG genes has resulted in distinct changes in how it is expressed in their testes.

evolutionary biology↗

bioRxiv2023

Transcriptional Regulation of the Alternative Sex Hormone-Binding Globulin Promoter by KLF4

AbstractIn most mammals the major site of sex hormone-binding globulin (SHBG) synthesis is the liver wherefrom it is secreted into the bloodstream and is the primary determinant of sex steroid access to target tissues. The minor site of SHBG synthesis is the testis and in lower mammals testicular SHBG has long been known to be synthesized and secreted by Sertoli cells. However, human testicular SHBG is expressed in developing germ cells from an upstream alternative promoter (altP-SHBG). Transcripts arising from this region comprise an alternative first exon (1A) with the resultant protein confined to the acrosomal compartment of the mature spermatozoa. I have dissected the regulatory components of the alternative SHBG promoter and identified motifs that are required for optimal transcriptional activity from this region. Transcriptional activity is driven by two CACCC elements that appear to be functionally redundant. The transcription factor KLF4 interacts with promoter the region spanning these elements in vivo. Knockdown of Klf4 results in decreased altP-SHBG activity, while Klf4 overexpression relieves the effects of knockdown. Based on their shared patterns of expression in vivo, I conclude that KLF4 is a transcriptional regulator of SHBG in male germ cells.

genetics↗

bioRxiv2023

Loss-of-function mutation in Omicron variants reduces spike protein expression and attenuates SARS-CoV-2 infection

SARS-CoV-2 Omicron variants emerged in 2022 with >30 novel amino acid mutations in the spike protein alone. While most studies focus on receptor binding domain changes, mutations in the C-terminus of S1 (CTS1), adjacent to the furin cleavage site, have largely been ignored. In this study, we examined three Omicron mutations in CTS1: H655Y, N679K, and P681H. Generating a SARS-CoV-2 triple mutant (YKH), we found that the mutant increased spike processing, consistent with prior reports for H655Y and P681H individually. Next, we generated a single N679K mutant, finding reduced viral replication in vitro and less disease in vivo. Mechanistically, the N679K mutant had reduced spike protein in purified virions compared to wild-type; spike protein decreases were further exacerbated in infected cell lysates. Importantly, exogenous spike expression also revealed that N679K reduced overall spike protein yield independent of infection. Although a loss-of-function mutation, transmission competition demonstrated that N679K had a replication advantage in the upper airway over wild-type SARS-CoV-2 in hamsters, potentially impacting transmissibility. Together, the data show that N679K reduces overall spike protein levels during Omicron infection, which has important implications for infection, immunity, and transmission.

microbiology↗

bioRxiv2020

Multi-parametric analysis of 58 SYNGAP1 variants reveal impacts on GTPase signaling, localization and protein stability

SYNGAP1 is a Ras and Rap GTPase with important roles in regulating excitatory synaptic plasticity. While many SYNGAP1 missense and nonsense mutations have been associated with intellectual disability, epilepsy, schizophrenia and autism spectrum disorder (ASD), there are many variants of unknown significance (VUS). In this report, we characterize 58 variants in nine assays that examine multiple aspects of SYNGAP1 function. Specifically, we used multiplex phospho-flow cytometry to measure the impact of variants on pERK, pGSK3{beta} and pCREB and high-content imaging to examine their subcellular localization. We find variants ranging from complete loss-of-function (LoF) to wildtype (WT)-like in their ability to regulate pERK and pGSK3{beta}, while all variants retain at least partial ability to regulate pCREB. Interestingly, our assays reveal that a high percentage of variants located within the disordered domain of unknown function that makes up the C-terminal half of SYNGAP1 exhibited LoF, compared to the more well studied catalytic domain. Moreover, we find protein instability to be a major contributor to dysfunction only for two missense variants both located within the catalytic domain. Using high-content imaging, we find variants with nuclear enrichment/exclusion and aberrant nuclear speckle localization. These variants are primarily located within the C2 domain known to mediate membrane lipid interactions. We find that mislocalization is distinct from altered catalytic activity, highlighting multiple independent molecular mechanisms underlying variant dysfunction. Our multidimensional dataset allows clustering of variants based on functional phenotypes and provides high-confidence pathogenicity classification.

genetics↗

bioRxiv2019

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PHTS, we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occurred across the protein, resulting in partial to complete loss of function (LoF), which was well correlated across models. However, assays were selectively sensitive to variants located in substrate binding and catalytic domains, which exhibited complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

genetics↗