Biology subjects

Chami, M.

Publications and source records attributed to Chami, M..

15 recordsLinked to original sources

bioRxiv2026

Uncoupling mycomembrane biogenesis from mycolic acid synthesis reveals a distinct role for mycoloyltransferases in mycobacterial cell division.

Bacteria of the order Mycobacteriales, including the genera Mycobacterium and Corynebacterium, possess a unique outer membrane, termed the mycomembrane, which is structurally and chemically distinct from the lipopolysaccharide-containing outer membrane of Gram-negative bacteria. A defining feature of the mycomembrane is its enrichment in mycolic acids, long-chain -branched, {beta}-hydroxylated fatty acids that occur as trehalose monomycolate (TMM), trehalose dimycolate (TDM), or are esterified to arabinogalactan, an unusual polymer that is itself covalently linked to peptidoglycan (PG). Mycoloyltransferases are Mycobacteriales-specific enzymes described to catalyze the transfer of mycolic acids from trehalose monomycolate (TMM) to various cell envelope acceptors, including trehalose and arabinogalactan. In several species, including Mycobacterium tuberculosis, these proteins are essential for viability; however, their occurrence as multiple paralogs with partially redundant functions has hindered the precise assignment of their cellular roles. Previously, we showed that Corynebacterium glutamicum remains viable in the absence of mycolic acids, and thus without a mycomembrane, following deletion of pks, the gene required for mycolic acid biosynthesis. Building on this finding, we systematically deleted all genes encoding mycoloyltransferases in C. glutamicum to further disclose their collective function in the cell. The resulting {Delta}myts mutant lacked arabinogalactan-bound mycolates and TDM, yet continued to synthesize TMM. Despite the high abundance of this major glycolipid, the {Delta}myts strain failed to assemble a mycomembrane and displayed pronounced cell aggregation. Unexpectedly, deletion of mycoloyltransferases also caused very severe defects in cell division and morphogenesis that are not observed in a {Delta}pks strain unable to synthesize mycolic acids. Together, these results demonstrate that mycoloyltransferases are essential for mycomembrane assembly but dispensable for TMM biosynthesis, and reveal an unexpected role for these enzymes in cell division that is independent of their canonical mycolic acid transfer activity. SIGNIFICANCEHow the mycomembrane is assembled and anchored to the cell wall remains a central question in Mycobacteriales, where this outer membrane is necessary for envelope integrity and intrinsic antibiotic resistance. By genetically separating mycolic acid synthesis from their incorporation into the outer membrane, we identify arabinogalactan-linked mycolates as the critical determinant for initiating membrane assembly. Unexpectedly, we also uncover a role for mycoloyltransferases beyond their canonical function in lipid metabolism, revealing a functional link with cell division. These findings point to a critical role of Mycoloyltransferases in the coordination between outer membrane biogenesis and bacterial cytokinesis.

microbiology↗

bioRxiv2025

Large extracellular vesicles containing mitochondria (EVMs) derived from Alzheimer disease cells harbor pathologic functional and molecular profiles and spread mitochondrial dysfunctions

In addition to small extracellular vesicles known as exosomes, cells release large extracellular vesicles containing mitochondria (EVMs). However, the molecular and functional characteristics of EVMs, as well as the impact of EVM secretion on the spreading of mitochondrial dysfunction between cells, remain unknown in the context of Alzheimers Disease (AD). Here, we provide an ultrastructural, biochemical, and functional characterization of EVMs isolated from cells expressing the amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). We identified differential proteomic and lipidomic signatures in APPswe-derived EVMs compared to control EVMs and revealed a specific proteomic profile in EVMs isolated from conditioned media of fibroblasts from AD patients at the prodromal stage of the disease. Our findings show that APP-C terminal fragments (APP-CTFs) pathogenic accumulation in cells potentiates EVM secretion through the budding of the plasma membrane. We lastly demonstrated that APP-CTFs loaded EVMs are active carriers of dysfunctional mitochondria that transfer mitochondrial pathology to naive control recipient cells.

neuroscience↗

bioRxiv2024

Hampered AMPK-ULK1 cascade in Alzheimer disease (AD) instigates mitochondria dysfunctions and AD-related alterations that are alleviated by metformin

BackgroundMitochondrial structure and function alterations are key pathological features in Alzheimers disease (AD) brains. The adenosine monophosphate-activated protein kinase (AMPK) and its downstream effector Unc-51 like autophagy activating kinase 1 (ULK1) represent a key node controlling mitochondria health, the alteration of which likely contribute to AD development. MethodsWe designed this study to investigate AMPK-ULK1 activation state in post-mortem human sporadic AD brains, in 3xTgAD mice that recapitulate most of human AD features, and in neuronal cells expressing the amyloid precursor protein with the familial Swedish mutation (APPswe). We examined the impact of the pharmacological and genetic modulation of AMPK-ULK1 cascade on mitochondria structure and functions in APPswe cells. We evaluated the potential beneficial impact of AMPK-ULK1 activation by Metformin (Met) on mitochondria defects, as well as on early- and late-stage AD-related alterations in vivo and ex vivo. ResultsAt first, we show that AMPK-ULK1 cascade is defective in murine and human AD brains as well as in APPswe cells. We then report that Met administration to 3xTgAD mice alleviates the alterations of neuronal mitochondria structure and function and we consolidate these results in cells using both pharmacological and genetic tools to modulate AMPK-ULK1 cascade. In mice brains, Met reduces the early accumulation of APP C-terminal fragments (APP-CTFs) as well as the amyloid beta (A{beta}) burden present in aged mice. Mechanistically, we show that Met increases the localization of APP-CTFs within cathepsin D-positive lysosomal compartments in vivo and enhances cathepsin D activity in vitro. The reduction of A{beta} load by Met occurs through an increased recruitment of Iba1+ cells to A{beta} plaques and an enhancement of the phagocytic activity of microglia. Accordingly, in symptomatic 3xTgAD mice, Met alleviates microgliosis and astrogliosis, modulates microglia morphology, reduces peripheral proinflammatory cytokines levels, and regulates the expression of a set of inflammatory genes. In addition, Met normalizes dendritic spines shape in organotypic hippocampal slice cultures modeling AD and improves learning performance of 3xTgAD mice. ConclusionsOur study demonstrates potential therapeutic benefits of targeting AMPK-ULK1 cascade to reverse both early and late AD-related alterations, deserving further investigation in fundamental research and in human clinical studies.

neuroscience↗

bioRxiv2023

Mitochondria alterations in fibroblasts from sporadic Alzheimer's disease (AD) patients correlate with AD-related clinical hallmarks

INTRODUCTIONMitochondria dysfunctions are key features in Alzheimers disease (AD). The occurrence of these disturbances in AD patients peripheral cells and their potential correlation with disease progression are under investigated. METHODSWe studied mitochondrial structure, function and mitophagy in fibroblasts including healthy volunteers and AD patients at the prodromal (AD-MCI) or demented (AD-D) stages. We carried out correlation studies with clinical cognitive scores, A{beta} plaques burden and the accumulation of peripheral amyloid precursor protein C-terminal fragments (APP-CTFs). RESULTSWe unveiled progressive alteration of mitochondria structure as well as specific mitochondrial dysfunctions signatures in AD-MCI and AD-D fibroblasts and show defective mitophagy and autophagy linked to impaired lysosomal activity in AD-D fibroblasts. We reported on significant correlations of a subset of these dysfunctions with cognitive decline, AD-related clinical hallmarks and peripheral APP-CTFs accumulation. DISCUSSIONThis study emphasizes the potential use of peripheral cells for investigating AD pathophysiology and likely diagnosis.

cell biology↗

bioRxiv2023

Molecular insights into atypical modes of beta-arrestin interaction with seven transmembrane receptors

{beta}-arrestins are multifunctional proteins that are critically involved in regulating spatio-temporal aspects of GPCR signaling. The interaction of {beta}-arrestins with GPCRs is typically conceptualized in terms of receptor activation and phosphorylation primarily in the carboxyl-terminus. Interestingly however, there are several GPCRs that harbor majority of phosphorylation sites in their 3rd intracellular loop (ICL3) instead of carboxyl-terminus but still robustly engage {beta}-arrestins. Moreover, there are several 7TMRs that are now characterized as intrinsically-biased, {beta}-arrestin-coupled receptors (ACRs) due to lack of functional G-protein-coupling but robust {beta}-arrestin binding leading to functional outcomes. The molecular basis of {beta}-arrestin interaction and activation upon binding to these types of 7TMRs is currently elusive, and it represents a major knowledge gap in our current understanding of this signaling system. Here, we present seven cryo-EM structures of {beta}-arrestins in basal state, activated by the muscarinic M2 receptor (M2R) through its ICL3, and a {beta}-arrestin-coupled receptor known as decoy D6 receptor (D6R). These structural snapshots combined with biochemical, cellular, and biophysical experiments including HDX-MS and MD simulation provide novel insights into the ability of {beta}-arrestins to preferentially select specific phosphorylation patterns in the receptors, and also illuminate the structural diversity in 7TMR-{beta}-arrestin interaction. Surprisingly, we also observe that the carboxyl-terminus of {beta}-arrestin2 but not {beta}-arrestin1 undergoes structural transition from a {beta}-strand to -helix upon activation by D6R, which may preclude the core-interaction with the activated receptor. Taken together, our study elucidates previously unappreciated aspects of 7TMR-{beta}-arrestin interaction, and provides important mechanistic clues about how the two isoforms of {beta}-arrestins can recognize and regulate a large repertoire of GPCRs.

biochemistry↗

bioRxiv2023

Structure-guided engineering of biased-agonism in the human niacin receptor via single amino acid substitution

The Hydroxycarboxylic acid receptor 2 (HCA2), also known as the niacin receptor or GPR109A, is a prototypical G protein-coupled receptor that plays a central role in the inhibition of lipolytic and atherogenic activities in our body. Interestingly, GPR109A activation also results in vasodilation that is linked to the side-effect of flushing associated with dyslipidemia drugs such as niacin. This receptor continues to be a key target for developing novel pharmacophores and lead compounds as potential therapeutics in dyslipidemia with minimized flushing response, however, the lack of structural insights into agonist-binding and receptor activation has limited the efforts. Here, we present five different cryo-EM structures of the GPR109A-G-protein complexes with the receptor bound to dyslipidemia drugs, niacin or acipimox, non-flushing agonists, MK6892 or GSK256073, and recently approved psoriasis drug, monomethyl fumarate (MMF). These structures allow us to visualize the binding mechanism of agonists with a conserved molecular interaction network, and elucidate the previously lacking molecular basis of receptor activation and transducer-coupling. Importantly, cellular pharmacology experiments, guided by the structural framework determined here, elucidate pathway-selective biased signaling elicited by the non-flushing agonists. Finally, taking lead from the structural insights, we successfully engineered receptor mutants via single amino acid substitutions that either fail to elicit agonist-induced transducer-coupling or exhibits G-protein signaling bias. Taken together, our study provides previously lacking structural framework to understand the agonist-binding and activation of GPR109A, and opens up the possibilities of structure-guided novel drug discovery targeting this therapeutically important receptor.

biochemistry↗

bioRxiv2023

Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the COVID-19 pandemic, already established methods were challenged by the large genome of SARS-CoV-2. Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity, using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment - harboring all heterologous sequences - viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

molecular biology↗

bioRxiv2023

Structural insights into ligand-recognition, activation, and signaling-bias at the complement C5a receptor, C5aR1

Activation of the complement cascade is a critical part of our innate immune response against invading pathogens, and it operates in a concerted fashion with the antibodies and phagocytic cells towards the clearance of pathogens. The complement peptide C5a, generated during the activation of complement cascade, is a potent inflammatory molecule, and increased levels of C5a are implicated in multiple inflammatory disorders including the advanced stages of COVID-19 pathophysiology. The proximal step in C5a-mediated cellular and physiological responses is its interaction with two different seven transmembrane receptors (7TMRs) known as C5aR1 and C5aR2. Despite a large body of functional data on C5a-C5aR1 interaction, direct visualization of their interaction at high-resolution is still lacking, and it represents a significant knowledge gap in our current understanding of complement receptor activation and signaling. Here, we present cryo-EM structures of C5aR1 activated by its natural agonist C5a, and a G-protein-biased synthetic peptide ligand C5apep, in complex with heterotrimeric G-proteins. The C5a-C5aR1 structure reveals the ligand binding interface involving the N-terminus and extracellular loops of the receptor, and we observe that C5a exhibits a significant conformational change upon its interaction with the receptor compared to the basal conformation. On the other hand, the structural details of C5apep-C5aR1 complex provide a molecular basis to rationalize the ability of peptides, designed based on the carboxyl-terminus sequence of C5a, to act as potent agonists of the receptor, and also the mechanism underlying their biased agonism. In addition, these structural snapshots also reveal activation-associated conformational changes in C5aR1 including outward movement of TM6 and a dramatic rotation of helix 8, and the interaction interface for G-protein-coupling. In summary, this study provides previously lacking molecular basis for the complement C5a recognition and activation of C5aR1, and it should facilitate structure-based discovery of novel lead molecules to target C5aR1 in inflammatory disorders.

biophysics↗

bioRxiv2022

Structural snapshots uncover a lock-and-key type conserved activation mechanism of β-arrestins by GPCRs

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a key determinant for the binding and activation of multifunctional regulatory proteins known as {beta}-arrestins ({beta}arrs). Although the primary sequence and phosphorylation pattern of GPCRs are poorly conserved, the downstream functional responses mediated by {beta}arrs such as receptor desensitization, endocytosis and signaling are broadly applicable across GPCRs. A conserved principle of {beta}arr activation, if any, upon their interaction with different GPCRs harboring divergent phosphorylation patterns remains to be visualized, and it represents a major knowledge gap in our current understanding of GPCR signaling and regulatory paradigms. Here, we present four structural snapshots of activated {beta}arrs, in complex with distinct phosphorylation patterns derived from the carboxyl-terminus of three different GPCRs, determined using cryogenic-electron microscopy (cryo-EM). These structures of activated {beta}arrs elucidate a "lock-and-key" type conserved mechanism of {beta}arr activation wherein a P-X-P-P phosphorylation pattern in GPCRs interacts with a spatially organized K-K-R-R-K-K sequence in the N-domain of {beta}arrs. Interestingly, the P-X-P-P pattern simultaneously engages multiple structural elements in {beta}arrs responsible for maintaining the basal conformation, and thereby, leads to efficient {beta}arr activation. The conserved nature of this lock-and-key mechanism is further illustrated by a comprehensive sequence analysis of the human GPCRome, and demonstrated in cellular context with targeted mutagenesis including "loss-of-function" and "gain-of-function" experiments with respect to {beta}arr activation measured by an intrabody-based conformational sensor. Taken together, our findings uncover previously lacking structural insights, which explain the ability of distinct GPCRs to activate {beta}arrs through a common mechanism, and a key missing link in the conceptual framework of GPCR-{beta}arr interaction and resulting functional outcomes.

biochemistry↗

bioRxiv2021

Apo and ligand-bound high resolution Cryo-EM structures of the human Kv3.1 reveal a novel binding site for positive modulators

Kv3 ion-channels constitute a class of functionally distinct voltage gated ion channels characterized by their ability to fire at a high frequency. Several disease relevant mutants, together with biological data, suggest the importance of this class of ion channels as drug targets for CNS disorders, and several drug discovery efforts have been reported. Despite the increasing interest for this class of ion channels, no structure of a Kv3 channel has been reported yet. We have determined the cryo-EM structure of Kv3.1 at 2.6 [A] resolution using full length wild type protein. When compared to known structures for potassium channels from other classes, a novel domain organization is observed with the cytoplasmic T1 domain, containing a well resolved Zinc site and displaying a rotation by 35{degrees}. This suggests a distinct cytoplasmic regulation mechanism for the Kv3.1 channel. A high resolution structure was obtained for Kv3.1 in complex with a novel positive modulator Lu AG00563. The structure reveals a novel ligand binding site for the Kv class of ion channels located between the voltage sensory domain and the channel pore, a region which constitutes a hotspot for disease causing mutations. The discovery of a novel binding site for a positive modulator of a voltage gated potassium channel could shed light on the mechanism of action for these small molecule potentiators. This finding could enable structure-based drug design on these targets with high therapeutic potential for the treatment of multiple CNS disorders.

biochemistry↗

bioRxiv2021

Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbBs are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the new class of inner membrane energy machinery formed by ExbB,ExbD and HasB.

microbiology↗

bioRxiv2021

Insight into Lipopolysaccharide Translocation by Cryo-EM structures of a LptDE Transporter in Complex with Pro-Macrobodies

Lipopolysaccharides (LPS) are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria 1. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex 2. Despite detailed insights from X-ray crystallography into the architecture of LptDE transporter complexes 3-5, no structure of a laterally open LptD transporter has been described, a transient state that occurs during LPS release 6. To facilitate the acquisition of hitherto unknown conformations we subjected LptDE of N. gonorrhoeae to cryo-EM analyses. In complex with newly designed rigid chaperones derived from nanobodies (Pro-Macrobodies, PMbs) we obtained a map of a partially opened LptDE transporter at 3.4 [A] resolution and in addition we captured a laterally fully opened LptDE complex from a subset of particles. Our work offers new insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a novel, highly rigid and widely applicable chaperone scaffold to enable structural biology of challenging protein targets.

biochemistry↗

bioRxiv2020

Cryo-EM structural studies of the agonist complexed human TRPV4 ion-channel reveals novel structural rearrangements resulting in an open-conformation

The human transient receptor potential vanilloid 4 (hTRPV4) ion channel plays a critical role in a variety of biological processes. Whilst the activation of hTRPV4 gating properties has been reported for a broad spectrum of stimuli, including synthetic 4-phorbols, the molecular basis of the activation is poorly understood. Here we report the novel cryo-EM structure of the hTRPV4 determined in the presence of the archetypical phorbol acid agonist, 4-PDD. Complementary mutagenesis experiments support the EM-identified binding site as well as allowing rationalization of disruptive mutants located outside of the 4-PDD binding site. This work represents the first structural information of hTRPV4 in a ligand-induced open conformation. Together, our data reveal the underlying molecular mechanisms resulting in the opening of the central pore and ion-channel activation and provide a structural template for designing inhibitors targeting the open-state conformation of hTRPV4.

molecular biology↗

bioRxiv2018

Retrieving High-Resolution Information from Disordered 2D Crystals by Single Particle Cryo-EM

Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are perfectly flat and highly ordered. In practice, such crystals are difficult to obtain. Available image unbending algorithms correct for disorder, but only perform well on images of non-tilted, flat crystals, while out-of-plane distortions are not addressed. Here, we present an approach that employs single-particle refinement procedures to locally unbend crystals in 3D. With this method, density maps of the MloK1 potassium channel with a resolution of 4 [A] were obtained from images of 2D crystals that do not diffract beyond 10 [A]. Furthermore, 3D classification allowed multiple structures to be resolved, revealing a series of MloK1 conformations within a single 2D crystal. This conformational heterogeneity explains the poor diffraction observed and is related to channel function. The approach is implemented in the FOCUS package.

biophysics↗

bioRxiv2017

Image processing techniques for high-resolution structure determination from badly ordered 2D crystals

2D electron crystallography can be used to study small membrane proteins in their native environment. Obtaining highly ordered 2D crystals is difficult and time-consuming. However, 2D crystals diffracting to only 10-12 [A] can be prepared relatively conveniently in most cases. We have developed image-processing algorithms allowing to generate a high resolution 3D structure from cryo-electron crystallography images of badly ordered crystals. These include movie-mode unbending, refinement over sub-tiles of the images in order to locally refine the sample tilt geometry; implementation of different CTF correction schemes; and an iterative method to apply known constraints in the real and reciprocal space to approximate amplitudes and phases in the so-called missing cone regions. These algorithms applied to a dataset of the potassium channel MloK1 show significant resolution improvements to approximately 5[A].\n\nAbbreviations

biophysics↗