Emergence of a short peptide based reductase via activation of the model hydride rich cofactor.

In extant biology, large and complex enzymes employ low molecular weight cofactors such as dihydronicotinamides as efficient hydride transfer agents and electron carriers for the regulation of critical metabolic processes. In absence of complex contemporary enzymes, these molecular cofactors are generally inefficient to facilitate any reactions on their own. Herein, we report short peptide-based amyloid nanotubes featuring exposed arrays of cationic and hydrophobic residues that can bind small molecular weak hydride transfer agents (NaBH4) to facilitate efficient reduction of ester substrates in water. In addition, the paracrystalline amyloid phases loaded with borohydrides demonstrate recyclability, substrate selectivity and controlled reduction and surpass the capabilities of standard reducing agent such as LiAlH4. The amyloid microphases and their collaboration with small molecular cofactors foreshadow the important roles that short peptide-based assemblies might have played in the emergence of protometabolism and biopolymer evolution in prebiotic earth.

Temporal (Dis)Assembly of Peptide Nanostructures Dictated by Native Multistep Catalytic Transformations.

Emergence of complex catalytic machinery via simple building blocks under non-equilibrium conditions can contribute toward the system level understanding of the extant biocatalytic reaction network that fuels metabolism. Herein, we report temporal (dis)assembly of peptide nanostructures in presence of a cofactor dictated by native multistep cascade transformations. The short peptide can form a dynamic covalent bond with the thermodynamically activated substrate and recruit cofactor hemin to access non-equilibrium catalytic nanostructures (positive feedback). The neighboring imidazole and hemin moieties in the assembled state rapidly converted the substrate to product(s) via a two-step cascade reaction (hydrolase-peroxidase like) that subsequently triggered the disassembly of the catalytic nanostructures (negative feedback). The feedback coupled reaction cycle involving intrinsic catalytic prowess of short peptides to realize the advanced trait of two-stage cascade degradation of a thermodynamically activated substrate foreshadows the complex non-equilibrium protometabolic networks that might have preceded the chemical emergence of life.

Exploitation of Catalytic Dyads by Short Peptide-Based Nanotubes for Enantioselective Covalent Catalysis.

Extant enzymes with precisely arranged multiple residues in their three-dimensional binding pockets are capable of exhibiting remarkable stereoselectivity towards a racemic mixture of substrates. However, how early protein folds that possibly featured short peptide fragments facilitated enantioselective catalytic transformations important for the emergence of homochirality still remains an intriguing open question. Herein, enantioselective hydrolysis was shown by short peptide-based nanotubes that could exploit multiple solvent-exposed residues to create chiral binding grooves to covalently interact and subsequently hydrolyse one enantiomer preferentially from a racemic pool. Single or double-site chiral mutations led to opposite but diminished and even complete loss of enantioselectivities, suggesting the critical roles of the binding enthalpies from the precise localization of the active site residues, despite the short sequence lengths. This work underpins the enantioselective catalytic prowess of short peptide-based folds and argues their possible role in the emergence of homochiral chemical inventory.

Temporal Self-Regulation of Mechanical Properties via Catalytic Amyloid Polymers of a Short Peptide.

We report a short peptide that accessed dynamic catalytic polymers to demonstrate four-stage (sol-gel-weak gel-strong gel) temporal self-regulation of its mechanical properties. The peptide exploited its intrinsic catalytic capabilities of manipulating C-C bonds (retro-aldolase-like) that resulted in a nonlinear variation in the catalytic rate. The seven-residue sequence exploited two lysines for binding and cleaving the thermodynamically activated substrate that subsequently led to the self-regulation of the mechanical strengths of the polymerized states as a function of time and reaction progress. Interestingly, the polymerization events were modulated by the different catalytic potentials of the two terminal lysines to cleave the substrate, covalently trap the electrophilic products, and subsequently control the mechanical properties of the system.

Emergence of Photomodulated Protometabolism by Short Peptide-Based Assemblies.

In the early Earth, rudimentary enzymes must have utilized the available light energy source to modulate protometabolic processes. Herein, we report the light-responsive C-C bond manipulation via short peptide-based assemblies bound to the photosensitive molecular cofactor (azo-based photoswitch) where the energy of the light source regulated the binding sites which subsequently modulated the retro-aldolase activity. In the presence of a continual source of high-energy photons, temporal realization of a catalytically more proficient state could be achieved under nonequilibrium conditions. Further, the hydrophobic surface of peptide assemblies facilitated the binding of an orthogonal molecular catalyst that showed augmented activity (promiscuous hydrolytic activity) upon binding. This latent activity was utilized for the in situ generation of light-sensitive cofactor that subsequently modulated the retro-aldolase activity, thus creating a reaction network.

Emergence of Catalytic Triad by Short Peptide Based Nanofibrillar Assemblies.

Through millions of years of the evolutionary journey, contemporary enzymes observed in extant metabolic pathways have evolved to become specialized, in contrast to their ancestors, which displayed promiscuous activities with wider substrate specificities. However, there remain critical gaps in our understanding of how these early enzymes could show such catalytic versatility despite lacking the complex three-dimensional folds of the existing modern-day enzymes. Herein, we report the emergence of a promiscuous catalytic triad by short amyloid peptide based nanofibers that access paracrystalline folds of ß-sheets to expose three residues (lysine, imidazole, and tyrosine) toward solvent. The ordered folded nanostructures could simultaneously catalyze two metabolically relevant chemical transformations via C-O and C-C bond manipulations, displaying both hydrolase and retro-aldolase-like activities. Further, the latent catalytic capabilities of the short peptide based promiscuous folds also helped in processing a cascade transformation, suggesting the important role they might have played in protometabolism and early evolutionary processes.

A ~24 kDa protein isolated from protein isolates of Hawaijar, popular fermented soy food of North-East India exhibited promising antidiabetic potential via stimulating PI3K/AKT/GLUT4 signaling pathway of muscle glucose metabolism.

The present study investigated the antidiabetic potential of protein isolates from Hawaijar (HPI), a popular fermented soybean food of North-East India. Treatment with HPI significantly upregulated glucose uptake, glucose utilization, glucose-6-phosphate, and stimulated PI3K/AKT/GLUT4 pathway in high-glucose (HG)-treated myotubes. Signal silencing studies demonstrated that knockdown of insulin-dependent signaling molecule (IR) but not insulin-independent signaling molecule (AMPK) significantly inhibited HPI-induced activation of PI3K/AKT/GLUT4 pathway and glucose uptake in HG-treated myotubes. SDS-PAGE and immunoblotting analyses of HPI showed the reduction and/or absence of various subunits of 7S and 11S globulin protein and appearance of new proteins compared to respective non-fermented soy protein isolates. Using various chromatographic techniques, the present study further isolated a single protein (ISP, ~24 kDa) from HPI as one of the bioactive principles with promising glucose utilization potential via stimulating PI3K/AKT/GLUT4 pathway in HG-treated cells. ISP treatment along with insulin significantly stimulated PI3K/AKT/GLUT4 pathway and glucose uptake compared to either insulin or ISP alone treated cells against HG exposure suggesting the insulin sensitizing effect of ISP. Furthermore, ISP supplementation significantly reduced metabolic markers linked with diabetes in high-fructose high-fat diet-fed animal model of type 2 diabetes. This study demonstrated a novel molecular mechanism underlying the promising antidiabetic potential of HPI.

Nonequilibrium Amyloid Polymers Exploit Dynamic Covalent Linkage to Temporally Control Charge-Selective Catalysis.

Extant proteins exploit thermodynamically activated negatively charged coenzymes and hydrotropes to temporally access mechanistically important conformations that regulate vital biological functions, from metabolic reactions to expression modulation. Herein, we show that a short amyloid peptide can bind to a small molecular coenzyme by exploiting reversible covalent linkage to polymerize and access catalytically proficient nonequilibrium amyloid microphases. Subsequent hydrolysis of the activated coenzyme leads to depolymerization, realizing a variance of the surface charge of the assembly as a function of time. Such temporal change of surface charge dynamically modulates catalytic activities of the transient assemblies as observed in highly evolved modern-day biocatalysts.

A popular fermented soybean food of Northeast India exerted promising antihyperglycemic potential via stimulating PI3K/AKT/AMPK/GLUT4 signaling pathways and regulating muscle glucose metabolism in type 2 diabetes.

This study examined the antidiabetic efficacy of popular fermented soybean foods (FSF) of Northeast (NE) India. Results showed that among different FSF, aqueous extract of Hawaijar (AEH), a traditional FSF of Manipur, NE India, significantly augmented glucose utilization in cultured myotubes treated with high glucose (HG, 25 mM). Furthermore, AEH also upregulated glucose uptake, glucose-6-phosphate level, and phopho-PI3K/phospho-AKT/phospho-AMPK/GLUT4 protein expression in HG-treated myotubes. In vivo studies demonstrated that AEH supplementation (50, 100, or 200 mg/kg body weight/day, oral gavaging, 16 weeks) reduced body weight, fasting blood glucose, glycated hemoglobin, insulin resistance, and glucose intolerance in rats fed with high-fat diet (HFD). AEH supplementation stimulated phopho-PI3K/phospho-AKT/phospho-AMPK/GLUT4 signaling cascades involved in glucose metabolism of muscle tissues in diabetic rats. Chemical profiling of AEH (SDS-PAGE, immunoblotting, and HRMS) suggests the possible role of bioactive proteins/peptides and isoflavones underlying the antihyperglycemic potential AEH. Results from this study will be helpful for developing food-based prophylactics/therapeutics in managing hyperglycemia. PRACTICAL APPLICATIONS: Fermented soybean foods are gaining acceptance due to multiple health benefits. This study for the first time reports the antidiabetic potential of Hawaijar, an indigenous fermented soybean food of North-East India. Higher abundance of bioactive compounds (isoflavones and proteins/peptides) in Hawaijar may be responsible for the alleviation of impaired glucose metabolism associated with diabetes. The findings may be helpful for the development of a novel therapeutic to achieve better control of hyperglycemia and improve the lives of the patient population with diabetes.

Systems chemistry of peptide-assemblies for biochemical transformations.

During the billions of years of the evolutionary journey, primitive polymers, involved in proto metabolic pathways with low catalytic activity, played critical roles in the emergence of modern enzymes with remarkable substrate specificity. The precise positioning of amino acid residues and the complex orchestrated interplay in the binding pockets of evolved enzymes promote covalent and non-covalent interactions to foster a diverse set of complex catalytic transformations. Recent efforts to emulate the structural and functional information of extant enzymes by minimal peptide based assemblies have attempted to provide a holistic approach that could help in discerning the prebiotic origins of catalytically active binding pockets of advanced proteins. In addition to the impressive sets of advanced biochemical transformations, catalytic promiscuity and cascade catalysis by such small molecule based dynamic systems can foreshadow the ancestral catalytic processes required for the onset of protometabolism. Looking beyond minimal systems that work close to equilibrium, catalytic systems and compartments under non-equilibrium conditions utilizing simple prebiotically relevant precursors have attempted to shed light on how bioenergetics played an essential role in chemical emergence of complex behaviour. Herein, we map out these recent works and progress where diverse sets of complex enzymatic transformations were demonstrated by utilizing minimal peptide based self-assembled systems. Further, we have attempted to cover the examples of peptide assemblies that could feature promiscuous activity and promote complex multistep cascade reaction networks. The review also covers a few recent examples of minimal transient catalytic assemblies under non-equilibrium conditions. This review attempts to provide a broad perspective for potentially programming functionality via rational selection of amino acid sequences leading towards minimal catalytic systems that resemble the traits of contemporary enzymes.

Antidiabetic potential of soy protein/peptide: A therapeutic insight.

Soybean (Glycine max) harbours high quality proteins which have been evident to exhibit therapeutic properties in alleviating many diseases including but not limited to diabetes and its related metabolic complications. Since diabetes is often manifested with hyperglycemia, impaired energy homeostasis and even low-grade chronic inflammation, plenty of information has raised the suggestion for soy protein supplementation in preventing and controlling these abnormalities. Moreover, clinical intervention studies have established a noteworthy correlation between soy protein intake and lower prevalence of diabetes. Besides soy protein, various soy-derived peptides also have been found to trigger antidiabetic response in different in vitro and in vivo models. Molecular mechanisms underlying the antidiabetic actions of soy protein and peptide have been predicted in many literatures. Results demonstrate that components of soy protein can act in diversified ways and modulate various cell signaling pathways to bring energy homeostasis and to regulate inflammatory parameters associated with diabetic pathophysiology. The main objective of the present review lies in a systemic understanding of antidiabetic role of soy protein and peptide in the context of impaired glucose and lipid metabolism, and inflammation.

Regulation of microtubule disassembly by spatially heterogeneous patterns of acetylation.

Microtubules (MTs) are bio-polymers, composed of tubulin proteins, involved in several functions such as cell division, transport of cargoes within cells, maintaining cellular structures etc. Their kinetics are often affected by chemical modifications on the filament known as Post Translational Modifications (PTMs). Acetylation is a PTM which occurs on the luminal surface of the MT lattice and has been observed to reduce the lateral interaction between tubulins on adjacent protofilaments. Depending on the properties of the acetylase enzyme αTAT1 and the structural features of MTs, the patterns of acetylation formed on MTs are observed to be quite diverse. In this study, we present a multi-protofilament model with spatially heterogeneous patterns of acetylation, and investigate how the local kinetic differences arising from heterogeneity affect the global kinetics of MT filaments. From the computational study we conclude that a filament with spatially uniform acetylation is least stable against disassembly, while ones with more clustered acetylation patterns may provide better resistance against disassembly. The increase in disassembly times for clustered pattern as compared to uniform pattern can be up to fifty percent for identical amounts of acetylation. Given that acetylated MTs affect several cellular functions as well as diseases such as cancer, our study indicates that spatial patterns of acetylation need to be focused on, apart from the overall amount of acetylation.

Conformational changes in the Ebola virus membrane fusion machine induced by pH, Ca2+, and receptor binding.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.

Covalent Catalysis by Cross ß Amyloid Nanotubes.

The binding pockets of extant enzymes feature precise positioning of amino acid residues that facilitate multiple complex transformations exploiting covalent and non-covalent interactions. Reversible covalent anchoring is extensively used as an efficient tool by Nature for activating modern enzymes such as esterases and dehydratases and also for proteins like opsins for the complex process of visual phototransduction. Here we construct paracrystalline amyloid surfaces through the self-propagation of short peptides which offer binding pockets exposed with arrays of imidazoles and lysines. As covalent catalysis is utilized by modern-day enzymes, these homogeneous amyloid nanotubes exploit Schiff imine formation via the exposed lysines to efficiently hydrolyze both activated and inactivated esters. Controls where lysines were mutated with charged residues accessed similar morphologies but did not augment the rate. The designed amyloid microphases thus foreshadow the generation of binding pockets of advanced proteins and have the potential to contribute to the development of functional materials.

Aldolase Cascade Facilitated by Self-Assembled Nanotubes from Short Peptide Amphiphiles.

Early evolution benefited from a complex network of reactions involving multiple C-C bond forming and breaking events that were critical for primitive metabolism. Nature gradually chose highly evolved and complex enzymes such as lyases to efficiently facilitate C-C bond formation and cleavage with remarkable substrate selectivity. Reported here is a lipidated short peptide which accesses a homogenous nanotubular morphology to efficiently catalyze C-C bond cleavage and formation. This system shows morphology-dependent catalytic rates, suggesting the formation of a binding pocket and registered enhancements in the presence of the hydrogen-bond donor tyrosine, which is exploited by extant aldolases. These assemblies showed excellent substrate selectivity and templated the formation of a specific adduct from a pool of possible adducts. The ability to catalyze metabolically relevant cascade transformations suggests the importance of such systems in early evolution.

Condensates of short peptides and ATP for the temporal regulation of cytochrome c activity.

Herein, we report the generation of simple condensates of short peptides with ATP, which are spatiotemporally formed under dissipative conditions created in presence of ATP-ase. These coacervates could imbibe cytochrome c and temporally modulate a redox reaction catalyzed by the entrapped protein, thus mimicking the advanced functional machinery of transient intercellular membraneless condensates of large proteins and RNA.

Gitelmans Syndrome- A Rare Cause of Recurrent Syncope.

Gitelman's syndrome, or congenital hypokalemic hypomagnesemic hypocalciuria with metabolic alkalosis, is widely described as a benign or milder variant of Barter's syndrome. It presents with variable clinical symptoms including tetanic episodes, muscle cramps, muscle paralysis, tingling numbness, perioral tingling sensation, salt craving and nocturia. This milder salt wasting syndrome can rarely cause significant ventricular arrhythmias and even death. Here, we report a case of 59 year old male who presented with history of recurrent syncope. He was found to have recurrent polymorphic VT with persistent hypokalemia and hypomagnesia. After extensive metabolic investigation, he was diagnosed as a case of Gitelman's syndrome. We report this case because of this rare malignant presentation of a seemingly benign syndrome.

Catalytic diversity in self-propagating peptide assemblies.

The protein-only infectious agents known as prions exist within cellular matrices as populations of assembled polypeptide phases ranging from particles to amyloid fibres. These phases appear to undergo Darwinian-like selection and propagation, yet remarkably little is known about their accessible chemical and biological functions. Here we construct simple peptides that assemble into well-defined amyloid phases and define paracrystalline surfaces able to catalyse specific enantioselective chemical reactions. Structural adjustments of individual amino acid residues predictably control both the assembled crystalline order and their accessible catalytic repertoire. Notably, the density and proximity of the extended arrays of enantioselective catalytic sites achieve template-directed polymerization of new polymers. These diverse amyloid templates can now be extended as dynamic self-propagating templates for the construction of even more complex functional materials.

Velocity distribution of a driven inelastic one-component Maxwell gas.

The nature of the velocity distribution of a driven granular gas, though well studied, is unknown as to whether it is universal or not, and, if universal, what it is. We determine the tails of the steady state velocity distribution of a driven inelastic Maxwell gas, which is a simple model of a granular gas where the rate of collision between particles is independent of the separation as well as the relative velocity. We show that the steady state velocity distribution is nonuniversal and depends strongly on the nature of driving. The asymptotic behavior of the velocity distribution is shown to be identical to that of a noninteracting model where the collisions between particles are ignored. For diffusive driving, where collisions with the wall are modeled by an additive noise, the tails of the velocity distribution is universal only if the noise distribution decays faster than exponential.

Theoretical estimates of exposure timescales of protein binding sites on DNA regulated by nucleosome kinetics.

It is being increasingly realized that nucleosome organization on DNA crucially regulates DNA-protein interactions and the resulting gene expression. While the spatial character of the nucleosome positioning on DNA has been experimentally and theoretically studied extensively, the temporal character is poorly understood. Accounting for ATPase activity and DNA-sequence effects on nucleosome kinetics, we develop a theoretical method to estimate the time of continuous exposure of binding sites of non-histone proteins (e.g. transcription factors and TATA binding proteins) along any genome. Applying the method to Saccharomyces cerevisiae, we show that the exposure timescales are determined by cooperative dynamics of multiple nucleosomes, and their behavior is often different from expectations based on static nucleosome occupancy. Examining exposure times in the promoters of GAL1 and PHO5, we show that our theoretical predictions are consistent with known experiments. We apply our method genome-wide and discover huge gene-to-gene variability of mean exposure times of TATA boxes and patches adjacent to TSS (+1 nucleosome region); the resulting timescale distributions have non-exponential tails.