Biology subjects

Krutyholowa, R.

Publications and source records attributed to Krutyholowa, R..

3 recordsLinked to original sources

bioRxiv2026

The small ORF (smORF)-encoded polypeptide MIRINE promotes neuronal differentiation in vertebrate brain development

The vertebrate brain expresses an extensive repertoire of long-noncoding RNAs (lncRNAs), an emerging source of small open reading frame (smORF)-encoded polypeptides (SEPs) whose functions remain largely unknown. Using ribosome profiling and proteomics of zebrafish and mouse brain, we identified hundreds of translated smORFs. We characterized MIRINE, a SEP of 83 amino acids conserved across 205 vertebrate species. MIRINE contains a transmembrane domain that targets it to the plasma membrane and intracellular vesicles, and is enriched in cortical and hippocampal neurons, with expression rising during neurogenesis. Downregulation of Mirine increased cell proliferation and impaired neuronal maturation in neuroblastoma cells, in mouse primary cortical neurons and cortex, and in the zebrafish forebrain, identifying MIRINE as a regulator of the balance between neuronal proliferation and differentiation. Mechanistically, MIRINE associates with the synaptic-vesicle protein Synaptogyrin-3 and related membrane proteins. Our findings define a conserved, membrane-associated SEP controlling neuronal maturation and illustrate the functional potential of brain SEPs.

biochemistry↗

bioRxiv2025

The EF-hand domain of MINDY3 is a Ubiquitin and RAD23 UBL-binding domain

The MINDY family of deubiquitinases (DUBs) are exemplified by their preference for cleaving K48-linked polyubiquitin. MINDY3 is architecturally distinct from other MINDY DUBs as its catalytic domain spans the entire length of the protein except for an atypical EF-hand insertion. We uncover this EF-hand (MINDY3EF-hand) to be a ubiquitin-binding domain with three distinct binding sites, enabling MINDY3 to bind and effectively cleave long polyubiquitin chains. Furthermore, the MINDY3EF-hand domain binds not only to polyubiquitin but also to the UBL domain of the proteasome shuttling and DNA repair factors RAD23A and RAD23B. The MINDY3EF-hand facilitates this interaction with RAD23s in cells and mediates MINDY3 recruitment to DNA damage sites, establishing this unique DUB as a potential regulator of cellular DNA damage responses. MINDY3 binds specifically to the UBL domain of RAD23s, and none of the other UBLs tested. The crystal structure of the MINDY3EF-hand:RAD23AUBL domain complex reveals the molecular basis for specificity. We find that MINDY3 can form a ternary complex with RAD23A/B and polyubiquitin, and our findings suggest a model wherein MINDY3 can deubiquitylate RAD23A/B-bound clients.

biochemistry↗

bioRxiv2023

Closure of the gamma-tubulin ring complex by CDK5RAP2 activates microtubule nucleation

Microtubule nucleation in cells is templated by the {gamma}-tubulin ring complex ({gamma}-TuRC), a 2.3 MDa multiprotein assembly concentrated at microtubule organizing centers (MTOCs). Current {gamma}-TuRC structures exhibit an open conformation that deviates from the geometry of /{beta}-tubulin in the microtubule, potentially explaining their low in vitro microtubule-nucleating activity. Several proteins have been proposed to activate the {gamma}-TuRC, but the mechanisms underlying activation are not known. Here, we isolated the porcine {gamma}-TuRC using CDK5RAP2s centrosomin motif 1 (CM1) and determined its structure with cryo-electron microscopy. 3D heterogeneity analysis revealed an unexpected conformation of the {gamma}-TuRC, in which five protein modules containing MZT2, GCP2, and CDK5RAP2 decorate the outer face of the holocomplex. These decorations drive a long-range constriction of the {gamma}-tubulin ring, bringing the GCP2/GCP3-rich core of the complex in close agreement with the architecture of a microtubule. A purified CDK5RAP2 fragment stimulated the microtubule nucleating-activity of the porcine {gamma}-TuRC as well as a reconstituted, CM1-free human complex in single molecule assays. Our results show that CDK5RAP2 activates the {gamma}-TuRC by promoting {gamma}-tubulin ring closure, providing a structural mechanism for the regulation of microtubule nucleation by CM1 motif proteins in mammals and revealing conformational transitions in {gamma}-tubulin that prime it for templating microtubule nucleation at MTOCs.

biochemistry↗