Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

Interplay of lipid head group and packing defects in driving amyloid-beta-mediated myelin-like model membrane deformation.

Accumulating evidence suggests that amyloid plaque-associated myelin lipid loss as a result of elevated amyloid burden might also contribute to Alzheimer's disease. The amyloid fibrils are closely associated with lipids under physiological conditions; however, the progression of membrane remodeling events leading to lipid-fibril assembly remains unknown. Here we first reconstitute the interaction of amyloid Beta 40 (Aß-40) with myelin-like model membrane and show that the binding of Aß-40 induces extensive tubulation. To look into the mechanism of membrane tubulation, we chose a set of membrane conditions varying in lipid packing density and net charge that allows us to dissect the contribution of lipid specificity of Aß-40 binding, aggregation kinetics, and subsequent changes in membrane parameters such as fluidity, diffusion, and compressibility modulus. We show that the binding of Aß-40 depends predominantly on the lipid packing defect densities and electrostatic interactions and results in rigidification of the myelin-like model membrane during the early phase of amyloid aggregation. Furthermore, elongation of Aß-40 into higher oligomeric and fibrillar species leads to eventual fluidization of the model membrane followed by extensive lipid membrane tubulation observed in the late phase. Taken together, our results capture mechanistic insights into snapshots of temporal dynamics of Aß-40-myelin-like model membrane interaction and demonstrate how short timescale, local phenomena of binding, and fibril-mediated load generation results in the consequent association of lipids with growing amyloid fibrils.

A Versatile Suspended Lipid Membrane System for Probing Membrane Remodeling and Disruption.

Artificial membrane systems can serve as models to investigate molecular mechanisms of different cellular processes, including transport, pore formation, and viral fusion. However, the current, such as SUVs, GUVs, and the supported lipid bilayers suffer from issues, namely high curvature, heterogeneity, and surface artefacts, respectively. Freestanding membranes provide a facile solution to these issues, but current systems developed by various groups use silicon or aluminum oxide wafers for fabrication that involves access to a dedicated nanolithography facility and high cost while conferring poor membrane stability. Here, we report the development, characterization and applications of an easy-to-fabricate suspended lipid bilayer (SULB) membrane platform leveraging commercial track-etched porous filters (PCTE) with defined microwell size. Our SULB system offers a platform to study the lipid composition-dependent structural and functional properties of membranes with exceptional stability. With dye entrapped in PCTE microwells by SULB, we show that sphingomyelin significantly augments the activity of pore-forming toxin, Cytolysin A (ClyA) and the pore formation induces lipid exchange between the bilayer leaflets. Further, we demonstrate high efficiency and rapid kinetics of membrane fusion by dengue virus in our SULB platform. Our suspended bilayer membrane mimetic offers a novel platform to investigate a large class of biomembrane interactions and processes.

Pore formation by pore forming membrane proteins towards infections.

Over the last 25 years, the biology of membrane proteins, including the PFPs-membranes interactions is seeking attention for the development of successful drug molecules against a number of infectious diseases. Pore forming toxins (PFTs), the largest family of PFPs are considered as a group of virulence factors produced in a large number of pathogenic systems which include streptococcus, pneumonia, Staphylococcus aureus, E. coli, Mycobacterium tuberculosis, group A and B streptococci, Corynebacterium diphtheria and many more. PFTs are generally utilized by the disease causing pathogens to disrupt the host first line of defense i.e. host cell membranes through pore formation strategy. Although, pore formation is the principal mode of action of the PFTs but they can have additional adverse effects on the hosts including immune evasion. Recently, structural investigation of different PFTs have imparted the molecular mechanistic insights into how PFTs get transformed from its inactive state to active toxic state. On the basis of their structural entity, PFTs have been classified in different types and their mode of actions alters in terms of pore formation and corresponding cellular toxicity. Although pathogen genome analysis can identify the probable PFTs depending upon their structural diversity, there are so many PFTs which utilize the local environmental conditions to generate their pore forming ability using a novel strategy which is known as "conformational switch" of a protein. This conformational switch is considered as characteristics of the phase shifting proteins which were often utilized by many pathogenic systems to protect them from the invaders through allosteric communication between distant regions of the protein. In this chapter, we discuss the structure function relationships of PFTs and how activity of PFTs varies with the change in the environmental conditions has been explored. Finally, we demonstrate these structural insights to develop therapeutic potential to treat the infections caused by multidrug resistant pathogens.

Membrane composition and lipid to protein ratio modulate amyloid kinetics of yeast prion protein.

Understanding of prion aggregation in a membrane environment may help to ameliorate neurodegenerative complications caused by the amyloid forms of prions. Here, we investigated the membrane binding-induced aggregation of yeast prion protein Sup35. Using the combination of fluorescence correlation spectroscopy (FCS) at single molecule resolution and other biophysical studies, we establish that lipid composition and lipid/protein ratio are key modulators of the aggregation kinetics of Sup35. In the presence of a zwitterionic membrane (DMPC), Sup35 exhibited novel biphasic aggregation kinetics at lipid/protein ratios ranging between 20 : 1 and 70 : 1 (termed here as the optimum lipid concentration, OLC). In ratios below (low lipid concentration, LLC) and above (ELC, excess lipid concentration) that range, the aggregation was found to be monophasic. In contrast, in the presence of negatively charged membranes, we did not observe any bi-phasic aggregation kinetics in the entire range of protein to lipid ratios. Our results provide a mechanistic description of the role that membrane concentration/composition-modulated aggregation may play in neurodegenerative diseases.

The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS model.

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, amyotrophic lateral sclerosis (ALS). Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid-induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a 'co-factor derived membrane association model' wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association-mediated toxic aggregation and survival time scale after ALS diagnosis.

Amyotrophic lateral sclerosis, or ALS, is an incurable neurodegenerative disease in which a person slowly loses specialized nerve cells that control voluntary movement. It is not fully understood what causes this fatal disease. However, it is suspected that clumps, or aggregates, of a protein called SOD1 in nerve cells may play a crucial role. More than 140 mutations in the gene for SOD1 have been linked to ALS, with varying degrees of severity. But it is still unclear how these mutations cause SOD1 aggregation or how different mutations influence the survival rate of the disease. The protein SOD1 contains a copper ion and a zinc ion, and it is possible that mutations that affect how these two ions bind to SOD1 influences the severity of the disease. To investigate this, Sannigrahi, Chowdhury, Das et al. genetically engineered mutants of the SOD1 protein which each contain only one metal ion. Experiments on these mutated proteins showed that the copper ion is responsible for the protein's role in neutralizing harmful reactive molecules, while the zinc ion stabilizes the protein against aggregation. Sannigrahi et al. found that when the zinc ion was removed, the SOD1 protein attached to a structure inside the cell called the mitochondria and formed toxic aggregates. Sannigrahi et al. then used these observations to build a computational model that incorporated different mutations that have been previously associated with ALS. The model suggests that mutations close to the site where zinc binds to the SOD1 protein increase disease severity and shorten survival time after diagnosis. This model was then experimentally validated using two disease variants of ALS that have mutations close to the sites where zinc or copper binds. These findings still need to be tested in animals and humans to see if these mechanisms hold true in a multicellular organism. This discovery could help design new ALS treatments that target the zinc binding site on SOD1 or disrupt the protein's interactions with the mitochondria.

Correlation between CNS Tuberculosis and the COVID-19 Pandemic: The Neurological and Therapeutic Insights.

The recent outbreak of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) from Wuhan, China, was caused by a single-stranded RNA virus which has kept the entire world stranded. The outbreak was first diagnosed with respiratory illness, but recent findings of acute necrotizing hemorrhage of brain, brain encephalopathy, and the presence of the virus in the cerebrospinal fluid (CSF) have unveiled its neuroinvasivness. Various clinical features related to the central nervous system (CNS) and peripheral nervous system (PNS) due to COVID-19 infection are now identified. We demonstrate here an apparent similarity in neurological disorders of COVID-19 with CNS tuberculosis, which suggests that some anti-tubercular drugs may be used as therapeutic agents against COVID-19 infection.

A Novel Cyclic Mobile Transporter Can Induce Apoptosis by Facilitating Chloride Anion Transport into Cells.

We report here the preparation of an aminoxy amide-based pseudopeptide-derived building block using furanoid sugar molecules. Through the cyclo-oligomerization reaction, we generate a hybrid triazole/aminoxy amide macrocycle using the as-prepared building block. The novel conformation of the macrocycle has been characterized using NMR and molecular modeling studies, which show a strong resemblance of our synthesized compound to d-,l-α-aminoxy acid-based cyclic peptides that contain uniform backbone chirality. We observe that the macrocycle can efficiently and selectively bind Cl- ion and transport Cl- ion across a lipid bilayer. 1H NMR anion binding studies suggest a coherent relationship between the acidity of aminoxy amide N-H and triazole C-H proton binding strength. Using time-based fluorescence assay, we show that the macrocycle acts as a mobile transporter and follows an antiport mechanism. Our synthesized macrocycle imposes cancer cell death by disrupting ionic homeostasis through Cl- ion transport. The macrocycle induced cytochrome c leakage and changes in mitochondrial membrane potential along with activation of family of caspases, suggesting that the cellular apoptosis occurs through a caspase-dependent intrinsic pathway. The present results suggest the possibility of using the macrocycle as a biological tool of high therapeutic value.

The bright and dark sides of protein conformational switches and the unifying forces of infections.

It is now established that a protein can switch between multiple conformations to enable altered functions. Several pathogens including SARS COV2 utilize context-dependent conformational switches of particular proteins to invade host membrane to establish infections. In this perspective, we first discuss the understanding of the conformational switch of a protein towards the productive infections as a dark side of nature. Next, the unexplored binary combination of the sequences of SARS COV2 spike protein and the similarity with diverse pathogen derived proteins have been discussed to obtain novel molecular insights into the process of infection.

Kinetoplastid Membrane Protein-11 Induces Pores in Anionic Phospholipid Membranes: Effect of Cholesterol.

Kinetoplastid membrane protein-11 (KMP-11), expressed in all stages of leishmanial life cycle, is considered a potential candidate for leishmaniasis vaccine. KMP-11 is found on the membrane surface of the parasite. Although the biological function of KMP-11 is unknown, we hypothesize from its sequence analysis that it may interact with the macrophage membrane and may influence the entry process of the parasite into the host cell. To validate this hypothesis, we have investigated the interaction of KMP-11 with unilamellar anionic phospholipid vesicles and explored its pore-forming activity. The decrease in negative ζ-potential of the vesicles and reduction in the fluorescence intensity of membrane-bound dye DiI C-18 suggest a strong association of KMP-11 with the membrane. The fluorescence leakage experiment as well as phase contrast microscopy shows direct evidence of KMP-11-induced pore formation in an anionic membrane. Incorporation of cholesterol into the membrane has been found to inhibit pore formation induced by KMP-11, suggesting an important role of cholesterol in leishmaniasis. Interestingly, vesicles containing only neutral phospholipid do not exhibit any tendency toward pore formation.

Conformational Switch Driven Membrane Pore Formation by Mycobacterium Secretory Protein MPT63 Induces Macrophage Cell Death.

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from ß-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native ß-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.

A Novel PEGylated Block Copolymer in New Age Therapeutics for Alzheimer's Disease.

The amyloid cascade hypothesis dealing with the senile plaques is until date thought to be one of the causative pathways leading to the pathophysiology of Alzheimer's disease (AD). Though many aggregation inhibitors of misfolded amyloid beta (Aß42) peptide have failed in clinical trials, there are some positive aspects of the designed therapeutic peptides for diseases involving proteinaceous aggregation. Here, we evaluated a smart design of side chain tripeptide (Leu-Val-Phe)-based polymeric inhibitor addressing the fundamental hydrophobic amino acid stretch "Lys-Leu-Val-Phe-Phe-Ala" (KLVFFA) of the Aß42 peptide. The in vitro analyses performed through the thioflavin T (ThT) fluorescence assay, infrared spectroscopy, isothermal calorimetry, cytotoxicity experiments, and so on evinced a promising path towards the development of new age AD therapeutics targeting the inhibition of misfolded Aß42 peptide fibrillization. The in silico simulations done contoured the mechanism of drug action of the present block copolymer as the competitive inhibition of aggregate-prone hydrophobic stretch of Aß42. Graphical abstract The production of misfolded Aß42 peptide from amyloid precursor protein initiates amyloidosis pathway which ends with the deposition of fibrils via the oligomerization and aggregation of Aß42 monomers. The side chain tripeptide-based PEGylated polymer targets these Aß42 monomers and oligomers inhibiting their aggregation. This block copolymer also binds and helps degrading the preformed fibrils of Aß42.

Development of a near infrared Au-Ag bimetallic nanocluster for ultrasensitive detection of toxic Pb2+ ions in vitro and inside cells.

Although the research activities pertaining to the synthesis of fluorescent noble metal nanoclusters (NCs) and their applications in biological optics have been growing, only limited information is available in the near IR (NIR) region. However, fluorescence spectroscopy and microscopy in the NIR region offer significant advantages over UV and visible wavelengths. In this manuscript, we demonstrate bio-mineralized synthesis of stable Au-Ag bimetallic NCs with tunable NIR fluorescence using bovine serum albumin (BSA) as a protein template. We also demonstrate its application in the detection of toxic heavy metal ions Pb2+ in vitro and inside cells. The tunability of the fluorescence emission between 680 nm and 815 nm is achieved by systematically varying the ratio of Au and Ag in the composite NCs. The bimetallic NCs when interacting with Pb2+ offered a large increase in fluorescence intensity, which enabled sensitive detection of Pb2+. We determined a limit of detection (LOD) of 96 nM for the detection of Pb2+ under in vitro conditions, which is significantly less than the safe level in drinking water. Its applicability has also been demonstrated successfully in real water samples collected from local water bodies.

Nanoparticle Induced Conformational Switch Between α-Helix and ß-Sheet Attenuates Immunogenic Response of MPT63.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from ß-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a ß-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

Interaction of KMP-11 with Phospholipid Membranes and Its Implications in Leishmaniasis: Effects of Single Tryptophan Mutations and Cholesterol.

KMP-11 is a small protein that is believed to control the overall bilayer pressure of the Leishmania parasite. Recent results have suggested that membrane binding and the presence of cholesterol affect the efficacy of Leishmanial infection, in which KMP-11 plays an important role. Nevertheless, there exists no systematic study of membrane interaction with KMP-11 either in the absence or presence of cholesterol. In this article, we investigated the interaction between KMP-11 and phospholipid membranes using an unsaturated (PC 18:1; 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)) and saturated (PC 12:0; 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) lipid as membrane mimics. Additionally, we studied the effect of cholesterol on the protein-membrane interaction. Steady-state as well as time-resolved fluorescence spectroscopy, isothermal titration calorimetry (ITC), and ζ-potential measurements were used for the determination of the binding constants for the wild-type (WT) and single-site tryptophan mutants. Single-site tryptophan mutants were designed to make sure that the tryptophan residues sample different surface exposures in different mutants. In the absence of cholesterol, the membrane-binding affinities of the partially exposed and buried tryptophan mutants (Y5W and Y48W, respectively) were found to be greater than those of the WT protein. In the presence of cholesterol, the binding constants of the WT and Y48W mutant were found to decrease with an increase in cholesterol concentration. This was in contrast to that in the Y5W and F77W mutants, in which the binding constants increased on adding cholesterol. The present study highlights the interplay among the conformational architecture of a protein, its interaction with the membrane, and membrane composition in modulating the survival of a Leishmania parasite inside host macrophages.