Biology subjects

Wass, A. B.

Publications and source records attributed to Wass, A. B..

5 recordsLinked to original sources

bioRxiv2025

Role of channels in the O2 permeability of murine red blood cells II. Morphological and proteomic studies

In this second of three papers, we examine red blood cell (RBC) morphometry and RBC-membrane proteomics from our laboratory mouse strain (C57BL/6Case). In paper #1, using stopped-flow absorbance spectroscopy to ascertain the rate constant for oxyhemoglobin (HbO2) deoxygenation (kHbO2), we find substantial kHbO2 reductions with (1) membrane-protein inhibitors p-chloromercuribenzenesulfonate (pCMBS) or 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS); (2) knockouts of aquaporin-1 (AQP1-KO), or Rhesus blood-group-associated A-glycoprotein (RhAG-KO), or double knockouts (dKO); or (3) inhibitor+dKO. In paper #3, reaction-diffusion mathematical modeling/simulations reveal that kHbO2 could fall secondary to slowed intracellular O2/HbO2/Hb diffusion. Here in paper #2, blood smears as well as still/video images and imaging flow cytometry (IFC) of living RBCs show that [~]97.5% to [~]98.6% of control (not drug-treated) cells are biconcave disks (BCDs) across all genotypes. Pretreatment with pCMBS raises non-BCD abundance to [~]8.7% for WT and [~]5.7% for dKO; for DIDS pretreatment, the figures are [~]41% and [~]21%, respectively. Modeling (paper #3) accommodates for these shape changes. Light-scattering flow cytometry shows no significant difference in RBC size or shape among genotypes. IFC reveals minor differences among genotypes in RBC major diameter (OMajor), which (along with mean corpuscular volume, paper #1) yields RBC thickness for simulations in paper #3. Label-free liquid chromatography/tandem mass spectrometry (LC/MS/MS) proteomic analyses of RBC plasma-membrane ghosts confirm the deletion of proteins targeted by our knockouts, and rule out changes in the 100 proteins of greatest inferred abundance. Thus, genetically induced changes in kHbO2 must reflect altered abundance of AQP1 and /or the Rh complex. Key PointsO_LIO2-offloading from red blood cells (RBCs) depends not only on membrane O2 permeability and oxyhemoglobin dissociation, but also on RBC size and shape. In this second of three papers, we use blood smears, still/video images of living RBCs, and imaging flow cytometry to examine morphometry of RBCs from paper #1. C_LIO_LIWe find that mouse RBCs of all genotypes--wild-type, aquaporin-1 knockout (AQP1-KO), Rhesus blood group-associated A-glycoprotein knockout (RhAG-KO), and double knockout--are dominantly biconcave discs, with [~]1.4% to [~]2.5% poikilocytosis (shape change, SC). Drug pre-treatment increases %SC. C_LIO_LIUsing label-free liquid chromatography/tandem mass spectrometry to assess apparent abundance of RBC-ghost proteins, we find no significant differences among genotypes for any of the [~]100 most abundant protein species except, as appropriate, AQP1, RhAG, or Rhesus blood group D antigen. C_LIO_LIThus, the substantial effects observed in paper #1 cannot be attributed to differences in morphometry or protein content. C_LI

physiology↗

bioRxiv2023

Virion-associated US28 rapidly modulates Akt activity to suppress HCMV lytic replication in monocytes

Establishing a non-productive quiescent/silent infection within monocytes is essential for spread of human cytomegalovirus (HCMV). Yet, how HCMV establishes a quiescent infection in monocytes remains unclear. US28 is a viral G protein-coupled receptor (GPCR) essential for silent infections within cells of the myeloid lineage. We found virion-associated US28 was rapidly delivered to monocytes, while de novo synthesized US28 was delayed for several days. A recombinant mutant virus lacking US28 (US28{Delta}) was unable to establish a quiescent infection, resulting in a fully productive lytic replication cycle. Mechanistically, viral entry of US28{Delta} phosphorylated Akt at both serine 473 (S473) and threonine 308 (T308), which contrasted with the site-specific phosphorylation of Akt at S473 following WT infection. Preventing Akt bi-phosphorylation prevented lytic replication of US28{Delta}, and ectopic expression of a constitutively phosphorylated Akt variant triggered lytic replication of WT infection. Our data demonstrate that virion-delivered US28 fine-tunes Akt activity to permit HCMV infection to enter a quiescent state following primary infection of monocytes.

microbiology↗

bioRxiv2020

Role of channels in the oxygen permeability of red blood cells

Many have believed that oxygen (O2) crosses red blood cell (RBC) membranes by dissolving in lipids that offer no resistance to diffusion. However, using stopped-flow (SF) analyses of hemoglobin (Hb) absorbance spectra during O2 off-loading from mouse RBCs, we now report that most O2 traverses membrane-protein channels. Two agents excluded from the RBC interior markedly slow O2 off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces inferred membrane O2 permeability (PMembrane) by [~]82%, and 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS), by [~]56%. Because neither likely produces these effects via membrane lipids, we examined RBCs from mice genetically deficient in aquaporin-1 (AQP1), the Rh complex (i.e., rhesus proteins RhAG + mRh), or both. The double knockout (dKO) reduces PMembrane by [~]55%, and pCMBS+dKO, by [~]91%. Proteomic analyses of RBC membranes, flow cytometry, hematology, and mathematical simulations rule out explanations involving other membrane proteins, RBC geometry, or extracellular unconvected fluid (EUF). By identifying the first two O2 channels and pointing to the existence of other O2 channel(s), all of which could be subject to physiological regulation and pharmacological intervention, our work represents a paradigm shift for O2 handling.

physiology↗

bioRxiv2020

Activator protein-1 transactivation of the major immediate early locus is a determinant of cytomegalovirus reactivation from latency

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that latently infects hematopoietic cells and has the ability to reactivate when triggered by immunological stress. This reactivation causes significant morbidity and mortality in immune-deficient patients, who are unable to control viral dissemination. While a competent immune system helps prevent clinically detectable viremia, a portrait of the factors that induce reactivation following the proper cues remains incomplete. Our understanding of the complex molecular mechanisms underlying latency and reactivation continue to evolve. We previously showed the HCMV-encoded G-protein coupled receptor US28 is expressed during latency and facilitates latent infection by attenuating the activator protein-1 (AP-1) transcription factor subunit, c-fos, expression and activity. We now show AP-1 is a critical component for HCMV reactivation. Pharmacological inhibition of c-fos significantly attenuates viral reactivation. In agreement, infection with a virus in which we disrupted the proximal AP-1 binding site in the major immediate early (MIE) enhancer results in inefficient reactivation compared to wild type. Concomitantly, AP-1 recruitment to the MIE enhancer is significantly decreased following reactivation of the mutant virus. Further, AP-1 is critical for de-repression of MIE-driven transcripts and downstream early and late genes, while immediate early genes from other loci remain unaffected. Our data also reveal MIE transcripts driven from the MIE promoter, the distal promoter, and the internal promoter, iP2, are dependent upon AP-1 recruitment, while iP1-driven transcripts are AP-1-independent. Collectively, our data demonstrate AP-1 binding to and activation of the MIE enhancer is a key molecular process controlling reactivation from latency. Significance StatementHuman cytomegalovirus (HCMV) is a common pathogen that infects the majority of the population for life. This infection poses little threat in immunologically healthy individuals, but can be fatal in people with compromised immune systems. Our understanding of the mechanisms underlying latency and reactivation remains incomplete. Here, we show the cellular transcription factor, AP-1, is a key to regulating HCMV reactivation. Our findings reveal AP-1 binding to the major immediate early enhancer/promoter is critical for switching this locus from one that is repressed during latency to one that is highly active following reactivation. Our work provides a novel mechanism HCMV exploits to reactivate, highlighting AP-1 as a potential target to prevent HCMV reactivation.

microbiology↗

bioRxiv2020

The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

microbiology↗