Biology subjects

Crynen, G.

Publications and source records attributed to Crynen, G..

7 recordsLinked to original sources

bioRxiv2024

Investigating cocaine- and abstinence-induced effects on astrocyte gene expression in the nucleus accumbens

In recent years, astrocytes have been increasingly implicated in cellular mechanisms of substance use disorders (SUD). Astrocytes are structurally altered following exposure to drugs of abuse; specifically, astrocytes within the nucleus accumbens (NAc) exhibit significantly decreased surface area, volume, and synaptic colocalization after operant self-administration of cocaine and extinction or protracted abstinence (45 days). However, the mechanisms that elicit these morphological modifications are unknown. The current study aims to elucidate the molecular modifications that lead to observed astrocyte structural changes in rats across cocaine abstinence using astrocyte-specific RiboTag and RNA-seq, as an unbiased, comprehensive approach to identify genes whose transcription or translation change within NAc astrocytes following cocaine self-administration and extended abstinence. Using this method, our data reveal cellular processes including cholesterol biosynthesis that are altered specifically by cocaine self-administration and abstinence, suggesting that astrocyte involvement in these processes is changed in cocaine-abstinent rats. Overall, the results of this study provide insight into astrocyte functional adaptations that occur due to cocaine exposure or during cocaine withdrawal, which may pinpoint further mechanisms that contribute to cocaine-seeking behavior.

neuroscience↗

bioRxiv2024

In vivo affinity maturation of the HIV-1 Env-binding domain of CD4

Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.

bioengineering↗

bioRxiv2023

In vivo affinity maturation of murine B cells reprogrammed to express human antibodies

CRISPR-edited murine B cells engineered to express human antibody variable chains proliferate, class switch, and secrete these antibodies in vaccinated mice. However, current strategies disrupt the heavy-chain locus, resulting in inefficient somatic hypermutation without functional affinity maturation. Here we show that recombined murine heavy- and kappa-variable genes can be directly and simultaneously overwritten, using Cas12a-mediated cuts at their 3-most J segments and 5 homology arms complementary to distal V segments. Cells edited in this way to express the HIV-1 broadly neutralizing antibodies 10-1074 or VRC26.25-y robustly hypermutated and generated potent neutralizing plasma in vaccinated recipient mice. 10-1074 variants isolated from these mice bound and neutralized HIV-1 envelope glycoprotein more efficiently than wild-type 10-1074 while maintaining or improving its already low polyreactivity and long in vivo half-life. We further validated this approach by generating substantially broader and more potent variants of the anti-SARS-CoV-2 antibodies ZCB11 and S309. Thus, B cells edited at their native loci affinity mature, facilitating development of broad, potent, and bioavailable antibodies and expanding the potential applications of engineered B cells.

bioengineering↗

bioRxiv2021

Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome in the cytoplasm of host cells, where cellular factors can antagonize or facilitate the virus life cycle. Here we leveraged proximity proteomics and conducted an siRNA screen to define the functional interactome of EBOV polymerase. As proof-of-principle, we validated two cellular mRNA decay factors from 35 identified host factors: eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1) and up-frameshift protein 1 (UPF1). Our data suggest that EBOV can subvert restrictions of cellular mRNA decay and repurpose both GSPT1 and UPF1 to promote viral replication. Treating EBOV-infected human hepatocytes with a drug candidate that targets GSPT1 for degradation significantly reduced viral RNA load and particle production. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

microbiology↗

bioRxiv2021

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host directed antivirals.

Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells, under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed an siRNA screen to identify those interactors that have functional roles in authentic LASV infection. As proof-of-principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet to be defined, host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses. Significance StatementLassa virus (LASV), the causative agent of Lassa fever (LF), represents an important public health problem in Western Africa. There is no FDA-approved therapeutic intervention to treat LF. Due to its limited genome coding capacity, LASV proteins are often multifunctional and orchestrate complex interactions with cellular factors to execute steps required to complete the viral life cycle. LASV polymerase is essential for replication and expression of the viral genome, and thus is an attractive target for antiviral intervention. Here we present the first host interactome of the LASV polymerase, which can guide identification of novel druggable host cellular targets for the development of cost-effective antiviral therapies for LF.

microbiology↗

bioRxiv2020

Rhes, a Striatal Enriched Protein, Regulates Post-Translational Small-Ubiquitin-like-Modifier (SUMO) Modification of Nuclear Proteins and Alters Gene Expression

Rhes (Ras homolog enriched in the striatum) is a multifunctional protein that orchestrates striatal toxicity, motor behaviors and abnormal movements associated with dopaminergic signaling, Huntington disease and Parkinson disease signaling in the striatum. Rhes engineers membranous tunneling nanotube-like structures and promotes intercellular protein and cargoes transport. Recent study revealed Rhes also regulates mitophagy via the Nix receptor. Despite these studies, the mechanisms through which Rhes mediates these diverse functions remains unclear. Rhes belongs to a small GTPase family member and consists of a unique C-terminal Small Ubiquitin-like Modifier (SUMO) E3-like domain that promotes the post-translational modification (PTM) of proteins with SUMO (SUMOylation) by promoting "cross-SUMOylation" of SUMO enzymes SUMO E1 (Aos1/Uba2) and SUMO E2 ligase (Ubc-9). However, the identity of the SUMO substrates of Rhes remains largely unknown. By combining high throughput interactome and SUMO proteomics we report that Rhes regulates the SUMOylation of nuclear proteins that are involved in the regulation of gene transcription. While Rhes has increased the SUMOylation of histone deacetylase 1 (HDAC1) and histone 2B, it had decreased the SUMOylation of heterogeneous nuclear ribonucleoprotein M (HNRNPM), protein polybromo-1 (PBRM1) and E3 SUMO-protein ligase (PIASy). We also found that Rhes itself is SUMOylated at 5 different lysine residues (K32, K110, K114, K120, K124 and K245). Furthermore, we found that Rhes regulates the expression of genes involved in cellular morphogenesis and differentiation in the striatum, in a SUMO-dependent manner. Our findings thus provide a previously undescribed role for Rhes in regulating SUMOylation of nuclear targets and in orchestrating striatal gene expression via the SUMOylation.

biochemistry↗

bioRxiv2019

RasGRP1 (CalDAG-GEF-II) Mediates L-DOPA-induced Dyskinesia in a Mouse Model of Parkinson Disease

The therapeutic benefits of L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson disease (PD) patients diminishes with the onset of abnormal involuntary movements (L-DOPA induced dyskinesia), a debilitating motor side effect. L-DOPA induced dyskinesia are due to altered dopaminergic signaling in the striatum, a brain region that controls motor and cognitive functions. However, the molecular mechanisms that promote L-DOPA-induced dyskinesia remain unclear. Here, we have reported that RasGRP1 (also known as CalDAG-GEF-II) physiologically mediated L-DOPA induced dyskinesia in a 6-hydroxy dopamine (6-OHDA) lesioned mouse model of PD. In this study, L-DOPA treatment rapidly upregulated RasGRP1 in the striatum. Our findings showed that RasGRP1 deleted mice (RasGRP1-/-) had drastically diminished L-DOPA-induced dyskinesia, and RasGRP1-/- mice did not interfere with the therapeutic benefits of L-DOPA. In terms of its mechanism, RasGRP1 mediates L-DOPA-induced extracellular regulated kinase (ERK), the mammalian target of rapamycin kinase (mTOR) and the cAMP/PKA pathway and binds directly with Ras-homolog-enriched in the brain (Rheb), which is a potent activator of mTOR, both in vitro and in the intact striatum. High-resolution tandem mass tag mass spectrometry analysis of striatal tissue revealed significant targets, such as phosphodiesterase (Pde1c), Pde2a, catechol-o-methyltransferase (comt), and glutamate decarboxylase 1 and 2 (Gad1 and Gad2), which are downstream regulators of RasGRP1 and are linked to L-DOPA-induced dyskinesia vulnerability. Collectively, the findings of this study demonstrated that RasGRP1 is a major regulator of L-DOPA-induced dyskinesia in the striatum. Drugs or gene-depletion strategies targeting RasGRP1 may offer novel therapeutic opportunities for preventing L-DOPA-induced dyskinesia in PD patients.

neuroscience↗