Integrative Proteomic and Pharmacological Analysis of Colon Cancer Reveals the Classical Lipogenic Pathway with Prognostic and Therapeutic Opportunities.

Despite recent advancements, the high mortality rate remains a concern in colon cancer (CAC). Identification of therapeutic markers could prove to be a great asset in CAC management. Multiple studies have reported hyperactivation of de novo lipogenesis (DNL), but its association with the pathology is unclear. This study aims to establish the importance as well as the prognostic and therapeutic potential of DNL in CAC. The key lipogenic enzymes fatty acid synthase along with ATP citrate lyase were quantified using an LC-MS/MS-based targeted proteomics approach in the samples along with the matched controls. The potential capacity of the proteins to distinguish between the tumor and controls was demonstrated using random forest-based class prediction analysis using the peptide intensities. Furthermore, in-depth proteomics of DNL inhibition in the CAC cell line revealed the significance of the pathway in proliferation and metastasis. DNL inhibition affected the major signaling pathways, including DNA repair, PI3K-AKT-mTOR pathway, membrane trafficking, proteasome, etc. The study revealed the upregulation of 26S proteasome machinery as a result of the treatment with subsequent induction of apoptosis. Again, in silico molecular docking-based drug repurposing was performed to find potential drug candidates. Furthermore, we have demonstrated that blocking DNL could be explored as a therapeutic option in CAC treatment.

Switching between blebbing and lamellipodia depends on the degree of non-muscle myosin II activity.

Cells can adopt both mesenchymal and amoeboid modes of migration through membrane protrusive activities, namely formation of lamellipodia and blebbing. How the molecular players control the transition between lamellipodia and blebs is yet to be explored. Here, we show that addition of the ROCK inhibitor Y27632 or low doses of blebbistatin, an inhibitor of non-muscle myosin II (NMII) ATPase activity and filament partitioning, induces blebbing to lamellipodia conversion (BLC), whereas addition of low doses of ML7, an inhibitor of myosin light chain kinase (MLCK), induces lamellipodia to blebbing conversion (LBC) in human MDA-MB-231 cells. Similarly, siRNA-mediated knockdown of ROCK and MLCK induces BLC and LBC, respectively. Interestingly, both blebs and lamellipodia membrane protrusions are able to maintain the ratio of phosphorylated to unphosphorylated regulatory light chain at cortices when MLCK and ROCK, respectively, are inhibited either pharmacologically or genetically, suggesting that MLCK and ROCK activities are interlinked in BLC and LBC. Such BLCs and LBCs are also inducible in other cell lines, including MCF7 and MCF10A. These studies reveal that the relative activity of ROCK and MLCK, which controls both the ATPase activity and filament-forming property of NMII, is a determining factor in whether a cell exhibits blebbing or lamellipodia.

Targeting mycobacterial membranes and membrane proteins: Progress and limitations.

Among the various bacterial infections, tuberculosis continues to hold center stage. Its causative agent, Mycobacterium tuberculosis, possesses robust defense mechanisms against most front-line antibiotic drugs and host responses due to their complex cell membranes with unique lipid molecules. It is now well-established that bacteria change their membrane composition to optimize their environment to survive and elude drug action. Thus targeting membrane or membrane components is a promising avenue for exploiting the chemical space focussed on developing novel membrane-centric anti-bacterial small molecules. These approaches are more effective, non-toxic, and can attenuate resistance phenotype. We present the relevance of targeting the mycobacterial membrane as a practical therapeutic approach. The review highlights the direct and indirect targeting of membrane structure and function. Direct membrane targeting agents cause perturbation in the membrane potential and can cause leakage of the cytoplasmic contents. In contrast, indirect membrane targeting agents disrupt the function of membrane-associated proteins involved in cell wall biosynthesis or energy production. We discuss the chronological chemical improvements in various scaffolds targeting specific membrane-associated protein targets, their clinical evaluation, and up-to-date account of their ''mechanisms of action, potency, selectivity'' and limitations. The sources of anti-TB drugs/inhibitors discussed in this work have emerged from target-based identification, cell-based phenotypic screening, drug repurposing, and natural products. We believe this review will inspire the exploration of uncharted chemical space for informing the development of new scaffolds that can inhibit novel mycobacterial membrane targets.

Lipidomic landscape in cancer: Actionable insights for membrane-based therapy and diagnoses.

Cancer cells display altered cellular lipid metabolism, including disruption in endogenous lipid synthesis, storage, and exogenous uptake for membrane biogenesis and functions. Altered lipid metabolism and, consequently, lipid composition impacts cellular function by affecting membrane structure and properties, such as fluidity, rigidity, membrane dynamics, and lateral organization. Herein, we provide an overview of lipid membranes and how their properties affect cellular functions. We also detail how the rewiring of lipid metabolism impacts the lipidomic landscape of cancer cell membranes and influences the characteristics of cancer cells. Furthermore, we discuss how the altered cancer lipidome provides cues for developing lipid-inspired innovative therapeutic and diagnostic strategies while improving our limited understanding of the role of lipids in cancer initiation and progression. We also present the arcade of membrane characterization techniques to cement their relevance in cancer diagnosis and monitoring of treatment response.

Lipidomic and Membrane Mechanical Signatures in Triple-Negative Breast Cancer: Scope for Membrane-Based Theranostics.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer associated with poor prognosis, higher grade, and a high rate of metastatic occurrence. Limited therapeutic interventions and the compounding issue of drug resistance in triple-negative breast cancer warrants the discovery of novel therapeutic targets and diagnostic modules. To this view, in addition to proteins, lipids also regulate cellular functions via the formation of membranes that modulate membrane protein function, diffusion, and their localization; thus, orchestrating signaling hot spots enriched in specific lipids/proteins on cell membranes. Lipid deregulation in cancer leads to reprogramming of the membrane dynamics and functions impacting cell proliferation, metabolism, and metastasis, providing exciting starting points for developing lipid-based approaches for treating TNBC. In this review, we provide a detailed account of specific lipidic changes in breast cancer, link the altered lipidome with membrane structure and mechanical properties, and describe how these are linked to subsequent downstream functions implicit in cancer progression, metastasis, and chemoresistance. At the fundamental level, we discuss how the lipid-centric findings in TNBC are providing cues for developing lipid-inspired theranostic strategies while bridging existing gaps in our understanding of the functional involvement of lipid membranes in cancer.

Ligand Induced CuII Transport Restricts Cancer and Mycobacterial Growth: Towards a Plug-and-Select Ion Channel Scaffold.

Synthetic channels with high ion selectivity are attractive drug targets for diseases involving ion dysregulation. Achieving selective transport of divalent ions is highly challenging due their high hydration energies. A small tripeptide amphiphilic scaffold installed with a pybox ligand selectively transports CuII ions across membranes. The peptide forms stable dimeric pores in the membrane and transports ions by a Cu2+ /H+ antiport mechanism. The ligand-induced excellent CuII selectivity as well as high membrane permeability of the peptide is exploited to promote cancer cell death. The peptide's ability to restrict mycobacterial growth serves as seeds to evolve antibacterial strategies centred on selectively modulating ion homeostasis in pathogens. This simple peptide can potentially function as a universal, yet versatile, scaffold wherein the ion selectivity can be precisely controlled by modifying the ligand at the C terminus.

Modulation of a host's cell membrane nano-environment by mycobacterial glycolipids: involvement of PI(4,5)P2 signaling lipid?

Virulence-associated glycolipids from Mycobacterium tuberculosis (Mtb) act as effector molecules during infection-in addition to proteins. Upon insertion, they alter the host cell's membrane properties modifying the host's functions to aid Mtb survival and disease course. Here we combine tether force experiments and microscopy to reveal previously unknown insights on the potential involvement of the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) lipid in the Mtb lipid-host interaction landscape. Our data shows that Mtb lipids, having different structural and chemical make-up, distinctly alter a host's PI(4,5)P2 membrane abundance/organization and PI(4,5)P2-actin colocalization, thus impacting the plasma membrane-cytoskeletal adhesion forces. Combined with our previous findings that underscore the role of exogenous Mtb lipids in remodeling host plasma membrane organization and mechanics, this work builds upon a lipid-centric view of tubercular infections. Dynamically changing a host's plasma membrane lipid content - in response to virulent lipids - might represent a so far unexplored mechanism invoked by Mtb to modulate the host cell's adhesive properties to escape immune surveillance. These findings will deepen our collective understanding of the functional role of Mtb lipids in hijacking the host cell processes amenable to pharmacological inhibition.

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.

Design and Expeditious Synthesis of Quinoline-Pyrene-Based Ratiometric Fluorescent Probes for Targeting Lysosomal pH.

Intracellular pH plays a significant role in many pathological and physiological processes. A series of quinoline-pyrene probes were synthesized in one-step fashion through an oxonium-ion-triggered alkyne carboamination sequence involving C-C, C-O and C-N bond formation for intracellular pH sensing. The quinoline-pyrenes showed significant red shifts at low pH. Fluorescence lifetime decay measurements of the probes showed decreases in lifetime at pH 4. The probes showed excellent selectivity in the presence of various potential interfering agents such as amino acids and cations/anions. Furthermore, the probes were found to show completely reversible emission behaviour in the window between pH 4 and 7. A morpholine-substituted quinoline-pyrene probe efficiently stained lysosomes with high Pearson correlation coefficients (0.86) with Lysotracker Deep Red DND-99 as a reference. A co-localization study of the probe with Lysotracker DND-99 showed selective intracellular targeting and a shift in fluorescence emission due to acidic lysosomal pH.

Various Facets of Pathogenic Lipids in Infectious Diseases: Exploring Virulent Lipid-Host Interactome and Their Druggability.

Lipids form an integral, structural, and functional part of all life forms. They play a significant role in various cellular processes such as membrane fusion, fission, endocytosis, protein trafficking, and protein functions. Interestingly, recent studies have revealed their more impactful and critical involvement in infectious diseases, starting with the manipulation of the host membrane to facilitate pathogenic entry. Thereafter, pathogens recruit specific host lipids for the maintenance of favorable intracellular niche to augment their survival and proliferation. In this review, we showcase the lipid-mediated host pathogen interplay in context of life-threatening viral and bacterial diseases including the recent SARS-CoV-2 infection. We evaluate the emergent lipid-centric approaches adopted by these pathogens, while delineating the alterations in the composition and organization of the cell membrane within the host, as well as the pathogen. Lastly, crucial nexus points in their interaction landscape for therapeutic interventions are identified. Lipids act as critical determinants of bacterial and viral pathogenesis by altering the host cell membrane structure and functions.

Lipidated Stapled Peptides Targeting the Acyl Binding Protein UNC119.

The acyl-binding UNC119 proteins mediate the activation and transport of various N-myristoylated proteins. In particular, UNC119a plays a crucial role in the completion of cytokinesis. Herein, we report the use of a lipidated peptide originating from the UNC119 binding partner Gnat1 as the basis for the design of lipidated, stabilized α-helical peptides that target UNC119a. By using the hydrocarbon peptide-stapling approach, cell-permeable binders of UNC119a were generated that induced the accumulation of cytokinetic and binucleated cells; this suggests UNC119a as a potential target for the inhibition of cytokinesis.

Membrane Dynamics in Health and Disease: Impact on Cellular Signalling.

Biological membranes display a staggering complexity of lipids and proteins orchestrating cellular functions. Superior analytical tools coupled with numerous functional cellular screens have enabled us to query their role in cellular signalling, trafficking, guiding protein structure and function-all of which rely on the dynamic membrane lipid properties indispensable for proper cellular functions. Alteration of these has led to emergence of various pathological conditions, thus opening an area of lipid-centric therapeutic approaches. This perspective is a short summary of the dynamic properties of membranes essential for proper cellular functions, dictating both protein and lipid functions, and mis-regulated in diseases. Towards the end, we focus on some challenges lying ahead and potential means to tackle the same, mainly underscored by multi-disciplinary approaches.

Chemical Genetics Reveals a Role of dCTP Pyrophosphatase 1 in Wnt Signaling.

Cell-based screening is a powerful approach to identify novel chemical modulators and biological components of relevant biological processes. The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis, and its deregulation plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members and regulators is of significant interest. By means of a cell-based assay monitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling. Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors. These findings indicate that dCTPP1 plays a role in regulation of Wnt/ß-catenin signaling most likely through a direct interaction with USP7.

Small Molecules Inspired by the Natural Product Withanolides as Potent Inhibitors of Wnt Signaling.

Wnt signaling is a fundamental pathway that drives embryonic development and is essential for stem cell maintenance and tissue homeostasis. Dysregulation of Wnt signaling is linked to various diseases, and a constitutively active Wnt pathway drives tumorigenesis. Thus, disruption of the Wnt response is deemed a promising strategy for cancer drug discovery. However, only few clinical drug candidates that target Wnt signaling are available so far, and new small-molecule modulators of Wnt-related processes are in high demand. Here we describe the synthesis of small molecules inspired by withanolide natural products by using a pregnenolone-derived ß-lactone as the key intermediate that was transformed into a δ-lactone appended to the D-ring of the steroidal scaffold. This natural-product-inspired compound library contained potent inhibitors of Wnt signaling that act upstream of the destruction complex to stabilize Axin in a tankyrase-independent manner.

Regulation of K-Ras4B Membrane Binding by Calmodulin.

K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell.

Novel approaches to map small molecule-target interactions.

The quest for small molecule perturbators of protein function or a given cellular process lies at the heart of chemical biology and pharmaceutical research. Bioactive compounds need to be extensively characterized in the context of the modulated protein(s) or process(es) in living systems to unravel and confirm their mode of action. A crucial step in this workflow is the identification of the molecular targets for these small molecules, for which a generic methodology is lacking. Herein we summarize recently developed approaches for target identification spurred by advances in omics techniques and chemo- and bioinformatics analysis.

Misplaced helix slows down ultrafast pressure-jump protein folding.

Using a newly developed microsecond pressure-jump apparatus, we monitor the refolding kinetics of the helix-stabilized five-helix bundle protein λ*YA, the Y22W/Q33Y/G46,48A mutant of λ-repressor fragment 6-85, from 3 µs to 5 ms after a 1,200-bar P-drop. In addition to a microsecond phase, we observe a slower 1.4-ms phase during refolding to the native state. Unlike temperature denaturation, pressure denaturation produces a highly reversible helix-coil-rich state. This difference highlights the importance of the denatured initial condition in folding experiments and leads us to assign a compact nonnative helical trap as the reason for slower P-jump-induced refolding. To complement the experiments, we performed over 50 µs of all-atom molecular dynamics P-drop refolding simulations with four different force fields. Two of the force fields yield compact nonnative states with misplaced α-helix content within a few microseconds of the P-drop. Our overall conclusion from experiment and simulation is that the pressure-denatured state of λ*YA contains mainly residual helix and little ß-sheet; following a fast P-drop, at least some λ*YA forms misplaced helical structure within microseconds. We hypothesize that nonnative helix at helix-turn interfaces traps the protein in compact nonnative conformations. These traps delay the folding of at least some of the population for 1.4 ms en route to the native state. Based on molecular dynamics, we predict specific mutations at the helix-turn interfaces that should speed up refolding from the pressure-denatured state, if this hypothesis is correct.

Effect of the N-Terminal Helix and Nucleotide Loading on the Membrane and Effector Binding of Arl2/3.

The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.

Pressure-A Gateway to Fundamental Insights into Protein Solvation, Dynamics, and Function.

Now that the centennial anniversary of the first report on pressure denaturation of proteins by Nobel Laureate P. W. Bridgman can be celebrated, this Review on the application of high pressure as a key variable for studying the energetics and interactions of proteins appears. We demonstrate that combined temperature-pressure-dependent studies help delineate the free-energy landscape of proteins and elucidate which features are essential in determining their stability. Pressure perturbation also serves as an important tool to explore fluctuations in proteins and reveal their conformational substates. From shaping the free-energy landscape of proteins themselves to that of their interactions, conformational fluctuations not only dictate a plethora of biological processes, but are also implicated in a number of debilitating diseases. Finally, the advantages of using pressure to explore biomolecular assemblies and modulate enzymatic reactions are discussed.