Dynamical Organization of Compositionally Distinct Inner and Outer Membrane Lipids of Mycobacteria.

Mycobacterium species, including Mycobacterium tuberculosis, employs atypical long (C60-90) and branched lipids to produce a complex cell wall and localizes these toward distinct spatial locations, inner membrane (IM) and outer membrane (OM), thus forming a robust permeability barrier. The properties and functional roles of these spatially orchestrated membrane platforms remain unknown. Herein, we report the distinctive lateral organization, fluidity, and lipid domain architecture of protein-free membranes reconstituted from IM and OM lipids in vitro from M. smegmatis (Msm) underscored by their lipid packing and lipid dynamics. We show that Msm OM, against common notion, is more dynamic and fluid compared with IM and reveal the role of cell wall-associated peptidoglycans and lipoarabinomannan on the Msm OM organization. Overall, these studies indicate that mycobacterial species may regulate their overall membrane functionality by regulating the synthesis of these complex arrays of lipids. Based on the structure-function relationship drawn here, documented alteration in the mycobacterial lipidome during cellular infection and/or drug treatment could reflect a mechanism to fine-tune M. tuberculosis membrane properties to its advantage. These findings are expected to inspire development of lipid-centric therapeutic approaches targeted toward its membrane.

Identification and Benchmarking of Myokinasib-II as a Selective and Potent Chemical Probe for Exploring MLCK1 Inhibition.

Deciphering the functional relevance of every protein is crucial to developing a better (patho)physiological understanding of human biology. The discovery and use of quality chemical probes propel exciting developments for developing drugs in therapeutic areas with unmet clinical needs. Myosin light-chain kinase (MLCK) serves as a possible therapeutic target in a plethora of diseases, including inflammatory diseases, cancer, etc. Recent years have seen a substantial increase in interest in exploring MLCK biology. However, there is only one widely used MLCK modulator, namely, ML-7, that too with a narrow working concentration window and high toxicity profile leading to limited insights. Herein, we report the identification of a potent and highly selective chemical probe, Myokinasib-II, from the synthesis and structure-activity relationship studies of a focused indotropane-based compound collection. Notably, it is structurally distinct from ML-7 and hence meets the need for an alternative inhibitor to study MLCK biology as per the recommended best practices. Moreover, our extensive benchmarking studies demonstrate that Myokinasib-II displays better potency, better selectivity profile, and no nonspecific interference in relevant assays as compared to other known MLCK inhibitors.

Residual Membrane Fluidity in Mycobacterial Cell Envelope Layers under Extreme Conditions Underlines Membrane-Centric Adaptation.

One of the routes for adaptation to extreme environments is via remodeling of cell membrane structure, composition, and biophysical properties rendering a functional membrane. Collective studies suggest some form of membrane feedback in mycobacterial species that harbor complex lipids within the outer and inner cell wall layers. Here, we study the homeostatic membrane landscape of mycobacteria in response to high hydrostatic pressure and temperature triggers using high pressure fluorescence, mass and infrared spectroscopies, NMR, SAXS, and molecular dynamics simulations. Our findings reveal that mycobacterial membrane possesses unique and lipid-specific pressure-induced signatures that attenuate progression to highly ordered phases. Both inner and outer membrane layers exhibit phase coexistence of nearly identical lipid phases keeping residual fluidity over a wide range of temperature and pressure, but with different sensitivities. Lipidomic analysis of bacteria grown under pressure revealed lipidome remodeling in terms of chain length, unsaturation, and specific long-chained characteristic mycobacterial lipids, rendering a fluid bacterial membrane. These findings could help understand how bacteria may adapt to a broad spectrum of harsh environments by modulating their lipidome to select lipids that enable the maintenance of a fluid functional cell envelope.

Biophysical Interaction Landscape of Mycobacterial Mycolic Acids and Phenolic Glycolipids with Host Macrophage Membranes.

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

The Emerging World of Membrane Vesicles: Functional Relevance, Theranostic Avenues and Tools for Investigating Membrane Function.

Lipids are essential components of cell membranes and govern various membrane functions. Lipid organization within membrane plane dictates recruitment of specific proteins and lipids into distinct nanoclusters that initiate cellular signaling while modulating protein and lipid functions. In addition, one of the most versatile function of lipids is the formation of diverse lipid membrane vesicles for regulating various cellular processes including intracellular trafficking of molecular cargo. In this review, we focus on the various kinds of membrane vesicles in eukaryotes and bacteria, their biogenesis, and their multifaceted functional roles in cellular communication, host-pathogen interactions and biotechnological applications. We elaborate on how their distinct lipid composition of membrane vesicles compared to parent cells enables early and non-invasive diagnosis of cancer and tuberculosis, while inspiring vaccine development and drug delivery platforms. Finally, we discuss the use of membrane vesicles as excellent tools for investigating membrane lateral organization and protein sorting, which is otherwise challenging but extremely crucial for normal cellular functioning. We present current limitations in this field and how the same could be addressed to propel a fundamental and technology-oriented future for extracellular membrane vesicles.

Small-Molecule Modulation of Lipid-Dependent Cellular Processes against Cancer: Fats on the Gunpoint.

Lipid cell membrane composed of various distinct lipids and proteins act as a platform to assemble various signaling complexes regulating innumerous cellular processes which are strongly downregulated or altered in cancer cells emphasizing the still-underestimated critical function of lipid biomolecules in cancer initiation and progression. In this review, we outline the current understanding of how membrane lipids act as signaling hot spots by generating distinct membrane microdomains called rafts to initiate various cellular processes and their modulation in cancer phenotypes. We elucidate tangible drug targets and pathways all amenable to small-molecule perturbation. Ranging from targeting membrane rafts organization/reorganization to rewiring lipid metabolism and lipid sorting in cancer, the work summarized here represents critical intervention points being attempted for lipid-based anticancer therapy and future directions.