Biology subjects

Ramos, N.

Publications and source records attributed to Ramos, N..

3 recordsLinked to original sources

bioRxiv2026

Quantitative and mutational analysis of soluble HIV-1 Vpu and calmodulin interactions

HIV-1 Vpu supports viral adaptation through host-protein interactions. Although mainly membrane-associated, we recently identified a soluble Vpu form that forms a stable complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially influencing Vpu trafficking. Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET). We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y/I33N mutant with substitutions of key residues in Vpu helix 1 and helices 1-to-2 loop having a role in the interaction with Ca2+-CaM. All Vpu variants were labeled at residue L42C. Ca2+-CaM was tagged with Cy5 at residue S39C. eFRET analysis of 100 nM Cy3-Vpu variants mixed with Cy5-Ca2+-CaM (in the range 100 nM-2.5 microM) revealed heterocomplexes formation with dissociation constants (Kd) and binding free energies (deltaG). FL Vpu-Ca2+-CaM showed highest stability (Kd ~74 nM, deltaG ~-9.7 kcal/mol), while the truncated C-terminal region and V22A/W23Y/I33N mutant formed weaker complexes with Ca2+-CaM (Kd ~182 nM and 800 nM, deltaG ~-9.2 kcal/mol and ~-8.3 kcal/mol). The reduced Vpu-Ca2+-CaM stability after disruption of binding sites in and near Vpu helix 1 may explain how this interaction is regulated, including through lipid competition that promotes Vpu membrane insertion. We propose that, at the membrane, hydrophobic helix 1 dissociates from Ca2+-CaM and inserts into the lipid bilayer, weakening the complex and releasing CaM. These findings clarify HIV-1 Vpu interactions with cellular components and may inform antiviral development.

biophysics↗

bioRxiv2022

Transposable elements drive the evolution of metazoan zinc finger genes

Cys2-His2 Zinc finger genes (ZNFs) form the largest family of transcription factors in metazoans. ZNF evolution is highly dynamic and characterized by the rapid expansion and contraction of numerous subfamilies across the animal phylogeny. The forces and mechanisms underlying rapid ZNF evolution remain poorly understood, but there is growing evidence that the targeting and repression of lineage-specific transposable elements (TEs) plays a major role in the diversification of the Kruppel-associated box ZNF (KZNF) subfamily, which predominates in tetrapod genomes. At present, it is unknown whether this function and co-evolutionary relationship is unique to KZNFs, or a broader feature of metazoan ZNFs. Here, we present evidence that genomic conflict with TEs has been a central driver in the diversification of ZNFs in animals. Sampling from more than 4000 animal genome assemblies, we show that the copy number of retroelements correlates with that of ZNFs across at least 750 million years of metazoan evolution, both within and between major taxonomic groups. Using computational predictions, we show that ZNFs preferentially bind TEs in a diverse set of representative animal species. We further investigate one of the most expansive ZNF subfamilies found in cyprinid fish, which are characterized by a conserved domain we dubbed the Fish N-terminal Zinc-finger associated (FiNZ) domain. FiNZ-ZNFs have dramatically expanded in several fish species, including the zebrafish in which we predict ~700 FiNZ-ZNF genes. Almost all are located on the long arm of chromosome 4, and recent duplicates are evolving adaptively under positive selection. Like mammalian KZNFs, the bulk of zebrafish FiNZ-ZNFs are expressed in waves at the onset of zygotic genome activation. Blocking FiNZ-ZNF translation using morpholinos during early zebrafish embryogenesis results in a global de-repression of young, transcriptionally active TEs, likely driven by the failure to establish heterochromatin over these elements. Together, these data suggest that ZNF diversification has been intimately connected to TE expansion throughout animal evolution and that families of ZNFs have been deployed independently in fish and mammals to repress TEs during early embryogenesis.

genomics↗

bioRxiv2022

Detection of IL23p40 via Positron Emission Tomography Visualized Inflammatory Bowel Disease

Background and aimsInflammatory bowel disease (IBD), which includes both Crohns Disease (CD) and ulcerative colitis (UC), is a relapsing inflammatory disease of the gastrointestinal (GI) tract. Long term chronic inflammatory conditions elevate patients risk for colorectal cancer (CRC). Currently, diagnosis requires endoscopy with biopsy. This procedure is invasive and requires bowel preparatory regimen, adding to patient burden. Interleukin 23 (IL23) plays a key role in inflammation especially in the pathogenesis of IBD and is an established therapeutic target. We propose that imaging of IL23 via immunopositron emission tomography (immunoPET) will potentially lead to a new non-invasive diagnostic approach. MethodsThe aim of the present study is to investigate the potential of immunoPET to image inflammation in a chemically induced mouse model of colitis using dextran sodium sulfate (DSS) by targeting IL23 via its p40 subunit with a 89Zr-radiolabeled -IL23p40 antibody. ResultsHigh uptake of the IL23p40 immunoPET agent in mice were displayed in DSS-administered mice, which correlated with increased IL23p40 present in sera. Competitive binding studies confirmed the specificity of the radiotracer for IL23p40 in the GI tract. ConclusionTaken together, these promising results set the stage for developing this radiotracer as an imaging biomarker for IBD diagnosis. Noninvasive imaging of IBD with IL23p40 immunoPET may help physicians in their treatment decisions for IBD management.

molecular biology↗