Biology subjects

Agrawal, R.

Publications and source records attributed to Agrawal, R..

24 records · Page 2Linked to original sources

bioRxiv2021

Allosteric Regulation of Human Plastins

Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis, and other cellular processes by organizing actin assemblies of strikingly different geometries as in aligned bundles and branched networks. We propose that this unique ability stems from an allosteric communication between plastins two actin-binding domains (ABD1/2) engaged in a tight spatial association. We found that although ABD1 binds actin first, ABD2 can bind to actin three orders of magnitude stronger if not inhibited by an equally strong allosteric engagement with ABD1. Binding of ABD1 to actin lessened the inhibition, enabling weak bundling within aligned bundles. A mutation mimicking physiologically relevant phosphorylation at the ABD1-ABD2 interface strongly reduced their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1-actin interface and enabled modeling of the plastin bridge to confirm domain separation in parallel bundles. The characteristic domain organization with a strong allosteric inhibition imposed by ABD1 on ABD2 allows plastins to tune cross-linking, contributing to the assembly and remodeling of actin assemblies with different morphological and functional properties defining the unique place of plastins in actin organization.

biochemistry↗

bioRxiv2021

Confined migration induces heterochromatin formation and alters chromatin accessibility

During migration, cells often squeeze through small constrictions, requiring extensive deformation. We hypothesized that the nuclear deformation associated with such confined migration could alter chromatin organization and function. Studying cells migrating through microfluidic devices that mimic interstitial spaces in vivo, we found that confined migration results in increased H3K9me3 and H3K27me3 heterochromatin marks that persist for several days. This "confined migration-induced heterochromatin" (CMiH) was distinct from heterochromatin formation during migration initiation. Confined migration predominantly decreased chromatin accessibility at intergenic regions near centromeres and telomeres, suggesting heterochromatin spreading from existing heterochromatin sites. Consistent with the overall decrease in chromatin accessibility, global transcription was decreased during confined migration. Intriguingly, we also identified increased accessibility at promoter regions of genes linked to chromatin silencing, tumor invasion, and DNA damage response. Inhibiting CMiH reduced migration speed, suggesting that CMiH promotes confined migration. Together, our findings indicate that confined migration induces chromatin changes that regulate cell migration and other cellular functions.

cell biology↗

bioRxiv2021

Single residue substitution in protamine 1 disrupts sperm genome packaging and embryonic development in mice

Conventional dogma presumes that protamine-mediated DNA compaction in sperm is achieved by passive electrostatics between DNA and the arginine-rich core of protamines. However, phylogenetic analysis reveals several non-arginine residues that are conserved within, but not across, species. The functional significance of these residues or post-translational modifications are poorly understood. Here, we investigated the functional role of K49, a rodent-specific lysine residue in mouse protamine 1 (P1) that is acetylated early in spermiogenesis and retained in sperm. In vivo, an alanine substitution (P1 K49A) results in ectopic histone retention, decreased sperm motility, decreased male fertility, and in zygotes, premature P1 removal from paternal chromatin. In vitro, the P1 K49A substitution decreases protamine-DNA binding and alters DNA compaction/decompaction kinetics. Hence, a single amino acid substitution outside the P1 arginine core is sufficient to profoundly alter protein function and developmental outcomes, suggesting that protamine non-arginine residues are essential to ensure reproductive fitness.

biochemistry↗

bioRxiv2021

Mapping the vocal circuitry of Alston's singing mouse with pseudorabies virus

Vocalizations, like many social displays, are often elaborate, rhythmically structured behaviors that are modulated by a complex combination of cues. Vocal motor patterns require close coordination of neural circuits governing the muscles of the larynx, jaw, and respiratory system. In the elaborate vocalization of Alstons singing mouse (Scotinomys teguina), for example, each note of its rapid, frequency-modulated trill is accompanied by equally rapid modulation of breath and gape. To elucidate the neural circuitry underlying this behavior, we introduced the polysynaptic retrograde neuronal tracer pseudorabies virus (PRV) into the cricothyroid and digastricus muscles, which control frequency modulation and jaw opening respectively. Each virus singly labels ipsilateral motoneurons (nucleus ambiguous for cricothyroid, and motor trigeminal nucleus for digastricus). We find that the two isogenic viruses heavily and bilaterally co-label neurons in the gigantocellular reticular formation, a putative central pattern generator. The viruses also show strong co-labeling in compartments of the midbrain including the ventrolateral periaqueductal grey and the parabrachial nucleus, two structures strongly implicated in vocalizations. In the forebrain, regions important to social cognition and energy balance both exhibit extensive co-labeling. This includes the paraventricular and arcuate nuclei of the hypothalamus, the lateral hypothalamus, preoptic area, extended amygdala, central amygdala, and the bed nucleus of the stria terminalis. Finally, we find doubly labeled neurons in M1 motor cortex previously described as laryngeal, as well as in the prelimbic cortex, which indicate these cortical regions play a role in vocal production. Although we observe some novel patterns of double-labelling, the progress of both viruses is broadly consistent with vertebrate-general patterns of vocal circuitry, as well as with circuit models derived from primate literature.

neuroscience↗

bioRxiv2021

Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis

A fundamental challenge in HIV eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral treatment (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV-1 is associated with down-regulation of the key H2S producing enzyme cystathionine-{gamma}-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-{kappa}B, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests the inclusion of an H2S donor in the current ART regimen to achieve a functional HIV-1 cure.

microbiology↗

bioRxiv2018

Structural insights into unique features of the human mitochondrial ribosome recycling

Mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing proteins that are essential for oxidative phosphorylation or energy (ATP) generation. Despite their proposed bacterial origin, the composition and structure of the human mitoribosome and its translational factors are dramatically different from their bacterial counterparts. The mammalian mitoribosome recycling factor (RRFmt) carries a mito-specific N-terminus extension (NTE), which is necessary for the function of RRFmt. Here we present a 3.7 [A] resolution cryo-EM structure of the human 55S mitoribosome-RRFmt complex, which reveals -helix and loop structures for a portion of the NTE that makes multiple mito-specific interactions with functionally critical regions of the mitoribosome. These include ribosomal RNAs segments that constitute the peptidyl transferase center (PTC), those that connect PTC with the GTPase-associated center, and with multiple mitoribosomal proteins. Our structure also reveals novel conformational changes in mitoribosome due to RRFmt binding. Together, these findings help understand the unique features of mitoribosome recycling.

biophysics↗