Biology subjects

Dubin, G.

Publications and source records attributed to Dubin, G..

8 recordsLinked to original sources

bioRxiv2025

Dynamics of complex genomic regions in giant genomes: MHC evolution in newts

Major Histocompatibility Complex (MHC) molecules are central to vertebrate adaptive immunity and MHC genes serve are key models in evolutionary genomics, offering insight into birth-and-death evolution, gene duplication, and the maintenance of genetic diversity. However, the organization and evolution of the MHC in species with giant genomes, such as salamanders, remain poorly understood. Here, we use comparative genomics, ontogenetic, tissue expression and polymorphism data across seven newt species to investigate MHC evolution in this group. Contrary to earlier suggestions of a massively expanded MHC in salamanders, we find that the core MHC region remains relatively compact, demonstrating that genome gigantism does not scale proportionally in this region. Our finding also challenges the model of coevolution between a single classical MHC-Ia gene and antigen processing genes (APGs), revealing instead several polymorphic and highly expressed putative MHC-Ia located at varying distances from the APGs. MHC-I genes exhibit lineage-specific duplications and signs of concerted evolution, resulting in poorly resolved phylogenies. In contrast, MHC-II genes are more conserved and exhibit extensive trans-species polymorphism. Expression and polymorphism patterns identify putative nonclassical MHC-Ib genes, likely repeatedly derived from MHC-Ia genes--paralleling patterns seen in mammals, but contrasting with the situation in fish and Xenopus frogs. In all seven species, some MHC-Ib genes show high relative expression during the larval stage but not at adulthood, suggesting a role in larval immunity. Our results underscore the importance of salamanders for understanding the evolution of complex regions in giant genomes and the architecture of the tetrapod MHC.

genomics↗

bioRxiv2024

Quantum Chemistry in a Pocket: A Multifaceted Tool to Link Structure and Activity

We introduce In-Pocket Analysis, a simple and efficient protein-ligand complex structure optimization algorithm. It provides structural biology and structure-based drug discovery with a much-needed tool for data curation and quantification analysis. In-pocket analysis removes unphysical tension from experimental structures, yielding high-quality atomic models. This includes deformed bonds, structural clashes, proton refinement, and solvent envelope. The algorithm is compatible with quantum methods or force fields, delivering precise calculations of binding energies and QSAR. Its applications include refinement of experimental structures, calculation of inter- and intramolecular energetics, scoring of docked poses, scaffold decoration/hopping, or building of explainable QSAR. For validation and benchmarking we used high-resolution crystal structures of the N-terminal domain of PEX14 with small-molecule inhibitors. PEX14 is a target candidate against Trypanosomiasis and Leishmaniasis. The application of our algorithm allowed for an explanation of unexpected binding poses, rationalization of binding energies, and affinities as well as precise quantification of solvent-mediated interactions. TABLE OF CONTENTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/590615v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@11f4173org.highwire.dtl.DTLVardef@1020358org.highwire.dtl.DTLVardef@12986ecorg.highwire.dtl.DTLVardef@1590617_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Noncanonical interactions and conformational dynamics in cargo-Pex5-Pex14 ternary complex for peroxisomal import

Peroxisomes are membrane enclosed organelles hosting diverse metabolic processes in eukaryotic cells. Having no protein synthetic abilities, peroxisomes import all required enzymes from the cytosol through a peroxin (pex) import system. Peroxisome targeting sequence 1 (PTS1)-tagged cargo is recognized by soluble receptor, Pex5. The cargo-Pex5 complex docks at the peroxisomal membrane translocon, composed of Pex14 and Pex13, facilitating translocation into the peroxisomal lumen. Despite its significance, the structural basis of this process is poorly understood. Here, we present a cryo-electron microscopy (cryo-EM) structure of cargo-Pex5-Pex14 ternary complex from Trypanosoma cruzi, the causative agent of human Chagas disease. The atomic model of the Pex5 (residues 327-653) bound to malate dehydrogenase (MDH, cargo) tetramer and Pex14NTD (residues 21-85) reveals dynamic motions and secondary interfaces. Specifically, we observe a swinging motion of Pex5 at the MDH-Pex5 interface. Additionally, the noncanonical contact surfaces are observed at the MDH-Pex5 and Pex5-Pex14 interfaces. Mutational analysis of the former interface indicates that it does not significantly enhance the affinity, underscoring the dynamic nature of cargo-Pex5 interactions. The latter interface constitutes an extended binding site of Pex14NTD over Pex5. We discuss the implications of these findings for understanding peroxisomal transport mechanisms. HIGHLIGHTSO_LIThe structure of the peroxisomal import ternary complex (MDH-Pex5TPR-Pex14NTD) is determined using cryo-EM. C_LIO_LIPex5 interface with the cargo (MDH) is characterized by significant conformational dynamics. C_LIO_LIThe extended binding site of Pex14NTD on Pex5 is revealed. C_LI

biochemistry↗

bioRxiv2022

Structural perspective on the design of selective DYRK1B inhibitors.

DYRK1B has been recently recognized as a critical therapeutic target in oncology and non-alcoholic fatty liver disease. However, the lack of structural information has constrained the development of selective inhibitors for DYRK1B. Here, we employed recombinant protein production, activity assays, and crystallization to elucidate the structure of DYRK1B. We present a crystal structure of DYRK1B in complex with a known inhibitor, AZ191. For comparative analysis, we provide the crystal structure of the closely related DYRK1A kinase in complex with AZ191. Our analysis identifies the exclusiveness of the binding site in the hinge region of DYRK1B, which is pivotal for selective inhibitor design. Quantum mechanical calculations reveal a notable difference in the accessibility of the catalytic lysine between DYRK1B and DYRK1A, offering avenues for distinguishing binders to these kinases. Our findings mark a significant advancement in the quest for specific DYRK1B inhibitors, potentially offering focused efficacy compared to the current dual-specificity inhibitors targeting both DYRK1B and DYRK1A.

biochemistry↗

bioRxiv2022

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During the RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10 mediated modulation remains unclear in the absence of nsp14 structure. Here we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10 bound structures explain the modulatory role of the co-factor protein. Further, the structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

microbiology↗

bioRxiv2022

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses protect their single-stranded RNA genome with a methylated cap during replication. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16 where nsp10 acts as a co-factor to both. Aditionally, nsp14 carries an exonuclease domain, which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins should be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex. This interaction is expected to encourage formation of mature capped viral mRNA, modulating the nsp14s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

microbiology↗

bioRxiv2021

Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses

SO_SCPLOWUMMARYC_SCPLOWThe COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

microbiology↗

bioRxiv2020

Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix (ECM), thus modulating the pericellular environment in physiology and excessively in pathologies like cancer. However, the interconnection between these groups of proteases remains elusive. To test this hypothesis, we have developed a peptide library-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences specifically. Initial assessment of the library identified a total of ten MMP activation domain sequences which were validated by Edman degradation. The CleavEx analysis revealed that membrane-type (MT) MMPs are likely targeted by KLK14 for activation. Correspondingly, commercially available proMT-MMPs, namely proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT MMPs were yielded and confirmed by Edman degradation. In addition, the productivity of proMMP activation was analyzed by gelatin zymography, which indicated the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts stably overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation within the ECM. As both, KLKs and MT-MMPs are implicated in cancer, the activation described herein may constitute an important factor in tumor progression and metastasis.

biochemistry↗