Biology subjects

Ruff, M.

Publications and source records attributed to Ruff, M..

7 recordsLinked to original sources

bioRxiv2023

The kinesin Kif21b regulates radial migration of cortical projection neurons through a noncanonical function on actin cytoskeleton.

Completion of neuronal migration is critical for brain development. Kif21b is a plus-end directed kinesin motor protein that promotes intracellular transport and controls microtubule dynamics in neurons. Here we report a physiological function of Kif21b during radial migration of projection neurons in the mouse developing cortex. In vivo analysis in mouse and live imaging on cultured slices demonstrate that Kif21b regulates the radial glia-guided locomotion of new-born neurons independently of its motility on microtubules. Unexpectedly we show that Kif21b directly binds and regulates the actin cytoskeleton both in vitro and in vivo in migratory neurons. We establish that Kif21b-mediated regulation of actin cytoskeleton dynamics influences branching and nucleokinesis during neuronal locomotion. Altogether, our results reveal atypical roles of Kif21b on the actin cytoskeleton during migration of cortical projection neurons.

neuroscience↗

bioRxiv2023

Biological and structural analysis of new potent Integrase-LEDGF allosteric HIV-1 inhibitors

LEDGF/p75 (LEDGF) main cellular cofactor of HIV-1 integrase (IN), acts as tethering factor to target integration of HIV in actively transcribed genes. Recently a class of IN inhibitors based on inhibition of LEDGF-IN interaction has been developed. We describe here a new series of IN-LEDGF allosteric inhibitors (INLAIs), with potent anti-HIV-1 activity in the one-digit nanomolar range. These compounds inhibited IN-LEDGF interaction while enhancing IN-IN aberrant multimerization by allosteric mechanism. Compounds of this series were fully active on HIV-1 mutants resistant to IN strand transfer inhibitors (INSTIs) and other class of anti-HIV drugs, confirming that they belong to a new class of antiretrovirals. These compounds displayed most potent antiretroviral activity at post-integration due to aberrant IN polymerization responsible of infectivity defect of viral particles produced. INLAI-resistant mutants were selected by dose escalation method and the most detrimental mutation found was T174I. The impact of these resistance mutations was analyzed by fold shift EC50 with regard to wild type virus and the replication capacity of mutated viruses were determined. Crystal structure of BDM-2, the lead compound of this series in complex with IN catalytic core domain (CCD) was elucidated. No antagonism was observed between BDM-2 and a panel of 16 antiretroviral drugs from different classes. In conclusion, the overall virologic profile of BDM-2 warrants the recently completed single ascending dose phase I trial (ClinicalTrials.gov Id: NCT03634085) and supports further clinical investigation for potential use in combination with other antiretroviral drugs. Author summaryIntegrase-LEDGF allosteric inhibitors are a new class of antiretrovirals recently developed that target the interaction of HIV-1 Integrase with its cellular cofactor LEDGF/p75 required for HIV-1 integration in actively transcribed genes. The great interest of developing such new class of antiretroviral is to add to the anti-HIV drug arsenal compounds that are fully active on resistant viruses to all other classes of drugs currently used in clinic. However, none of these inhibitors are currently in clinical use or in late clinical trials. Here we describe a series of highly potent Integrase allosteric inhibitors that can be considered as precursors of a new class of antiretroviral drugs, with fully conserved activity on viruses resistant to currently used anti-HIV drugs, and a lead compound that has no antagonism with a large panel of present anti-HIV drugs and that was successfully validated in a phase I clinical trial.

microbiology↗

bioRxiv2022

Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA Binding Proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically-loaded focal adhesions by modulation of integrin signaling and local delivery of {beta}-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks.

cell biology↗

bioRxiv2022

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome

Retroviral integration into cell chromatin requires the formation of integrase-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes. To further study this mechanism we developed an alphaLISA approach using the prototype foamy virus (PFV) intasome and human nucleosome. This system allowed us to monitor the association between both partners and investigate the protein/protein and protein/DNA interactions engaged in the association with chromatin. Using this approach, we next screened the chemical OncoSET library and selected small molecules that could modulate the intasome/nucleosome complex. Molecules were selected as acting either on the DNA topology within the nucleosome or on the integrase/histone tail interactions. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, structural and cellular approaches. These drugs were shown to inhibit PFV and HIV-1 integration in vitro as well as HIV-1 infection in primary PBMCs cells. Our work provides new information about intasome-nucleosome interaction determinants and paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring.

microbiology↗

bioRxiv2021

The HIV-1 Integrase C-Terminal domain induces TAR RNA structural changes promoting Tat binding.

Recent evidence indicated that HIV-1 Integrase (IN) binds genomic viral RNA (gRNA) playing a critical role in viral particle morphogenesis and gRNA stability in host cells. Combining biophysical and biochemical approaches we show that the C-terminal flexible 18-residues tail of IN acts as a sensor of the peculiar apical structure of trans-activation response element RNA (TAR), directly interacting with its hexaloop. We highlighted how the whole IN C-terminal domain, once bound to TAR, can change its structure assisting the binding of Tat, the HIV trans-activator protein, which finally displaces IN from TAR. Our results are consistent with the emerging role of IN in early stage of proviral transcription and suggest new steps of HIV-1 life cycle that can be considered as therapeutic targets.

biochemistry↗

bioRxiv2020

Analysis of the intrinsic chromatin binding property of HIV-1 integrase and its regulation by LEDGF/p75 using human chromosomes spreads

Retroviral integration requires the stable insertion of the viral genome into the host chromosomes. During this process, the functional integration complex must associate with cellular chromatin via the interaction between retroviral integrase and nucleosomes. The final association between the HIV-1 integration complex and the nucleosomal target DNA remains unclear and may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To date, there is no experimental system allowing the direct monitoring of this protein association with chromatin to depict the molecular mechanism of this process fully. To investigate this and understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase further, we used both biochemical approaches and an unedited chromosome-binding assays. Our study revealed that retroviral IN has an intrinsic ability to bind and recognize specific chromatin regions even in the absence of its cofactor. We also showed that this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromatin regions. Using these approaches, we also better determined the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. In addition to supporting a chromatin-binding function of the integrase protein acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate, our work precisely elucidates the mechanism of action of LEDGF/p75 in this crucial integration step.

microbiology↗

bioRxiv2020

Genetically flexible but conserved: a new essential motif in the C-ter domain of HIV-1 group M integrases

Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. IMPORTANCEIntensive studies on HIV-1 have revealed its extraordinary ability to adapt to environmental and immunological challenges, an ability that is also at the basis of antiviral treatments escape. Here, by deconvoluting the different roles of the viral integrase in the various steps of the infectious cycle, we report how the existence of alternative equally efficient structural arrangements for carrying out one function opens on the possibility of adapting to the optimisation of further functionalities exerted by the same protein. Such property provides an asset to increase the efficiency of the infectious process. On the other hand, though, the identification of this new motif provides a potential target for interfering simultaneously with multiple functions of the protein.

molecular biology↗