Residue Peptide Research Articles

De novo design of a stapled peptide targeting SARS-CoV-2 spike protein receptor-binding domain.

Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have low rates of vaccination and new variants with mutations in the viral spike protein have reduced the effectiveness of most available vaccines and treatments. There is an urgent need for a drug to cure this disease and prevent infection. The SARS-CoV-2 virus enters the host cell through protein-protein interaction between the virus's spike protein and the host's angiotensin converting enzyme (ACE2). Using protein design software and molecular dynamics simulations, we have designed a 17-residue peptide (pep39), that binds to the spike protein receptor-binding domain (RBD) and blocks interaction of spike protein with ACE2. We have confirmed the binding activity of the designed peptide for the original spike protein and the delta variant spike protein using micro-cantilever and bio-layer interferometry (BLI) based methods. We also confirmed that pep39 strongly inhibits SARS-CoV-2 virus replication in Vero E6 cells. Taken together these data suggest that a newly designed spike protein RBD blocking peptide pep39 has a potential as a SARS-CoV-2 virus inhibitor.

Prediction of Quality-control Degradation Signals in Yeast Proteins

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin–proteasome system. This is achieved via recognition of so-called degradation motifs—degrons—that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

A Frog-Derived Cathelicidin Peptide with Dual Antimicrobial and Immunomodulatory Activities Effectively Ameliorates Staphylococcus aureus-Induced Peritonitis in Mice.

As antimicrobial resistance poses an increasing threat to public health, it is urgent to develop new antimicrobial agents. In this paper, we identify a novel 30-residue peptide (Nv-CATH, NCNFLCKVKQRLRSVSSTSHIGMAIPRPRG) from the skin of the frog Nanorana ventripunctata, which belongs to the cathelicidin family. Nv-CATH exhibited broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. Nv-CATH significantly protected mice from lethal infections caused by Staphylococcus aureus. Furthermore, the peptide suppressed excessive and harmful inflammatory responses by repressing the production of NO, IL-6, TNF-α, and IL-1β. The NF-κB-NLRP3 and MAPK inflammatory signaling pathways were involved in the protection in vitro and in vivo. Nv-CATH also modulated macrophage/monocyte and neutrophil trafficking to the infection site by stimulating CXCL1, CXCL2, and CCL2 production in macrophages. Nv-CATH augmented immunocyte-mediated bacterial killing by modestly promoting neutrophils' phagocytosis and PMA-induced NET formation. Thus, Nv-CATH protects mice against bacterial infection by antimicrobial-immunomodulatory duality. The combination of these two characteristics makes Nv-CATH a promising molecule template for the development of novel antimicrobial and antibiotic-resistant agents.

DDDR-19. NEXT-GENERATION PI3K INHIBITOR FOR GLIOBLASTOMA TREATMENT

Abstract High-grade gliomas are especially problematic because of the limited treatment options available to patients. The phosphoinositide 3-kinase (PI3K) pathway has become a significant research target within the field of cancer drug discovery due to the pathway’s promotion of cell proliferation and inhibition of apoptosis. While many new PI3K inhibitory medications show promise in preventing cancer growth, many are toxic because they inhibit multiple PI3K catalytic subunit isoforms making these drugs ineffective treatment options. Our novel peptide drug, Selectide-18, overcomes this toxicity by specifically targeting the p110β isoform of PI3K, which was shown to be the most impactful isoform in many cancer types, including several high-grade gliomas. We designed Selectide-18 after discovering an 18-residue motif unique to p110β. Selectide-18 is a mimetic peptide modeled after this motif. Our in silico analyses show that our drug works by binding to the PI3K regulatory subunit, p85α, thus preventing the binding of catalytic subunit p110β and leaving PI3K inactive. These findings were supported by coimmunoprecipitation. Using a PI3K cell-free kinase assay, we confirmed that our drug specifically targets p110β and does not inhibit p110α, p110δ, or p110γ. The IC50 of Selectide-18 was determined to be approximately 100nM using the same PI3K cell-free kinase assay. We tested several drugs in an MTS viability assay, including Selectide-18, a scrambled 18-residue negative control peptide, and PI3K inhibitor GSK2636771 to compare levels of cancer cell viability. Selectide-18 successfully killed glioblastoma cells within lines with high p110β activity. The drug had no effect in cell lines with low p110β activity. In conclusion, Selectide-18 is explicitly tailored for p110β-high gliomas and has shown promise to treat high-grade glioma effectively. We believe this model of peptide drug can usher in a new generation of p110β specific PI3K inhibitors.

Cutinase fused with C-terminal residues of α-synuclein improves polyethylene terephthalate degradation by enhancing the substrate binding

Environmental-friendly treatment of poly (ethylene terephthalate) (PET) is important for the circular plastic economy, and enzymatic degradation provided a promising approach. However, enzyme binding toward the PET surface is a rate-limiting factor in PET hydrolysis. Thus, a 16-residue peptide (αSP) from the C-terminal sequence of α-synuclein was used to fuse to the C-terminal of a variant (ICCG) of the leaf-branch compost cutinase, and supposed to serve as an anchor peptide for PET degradation. The fusion enzyme (ICCG-αSP) was characterized to keep the same secondary and tertiary structures as ICCG, but enhanced enzyme binding onto bis(2-hydroxyethyl) terephthalate and PET, respectively. Moreover, αSP-fusion enhanced enzyme expression by over 50%, and increased the catalytic efficiency of p-nitrophenyl butyrate by 20% without compromising the enzyme stability. For PET degradation, ICCG-αSP was 3.89 times more active than ICCG by 3-h reaction at 50 °C, and exhibited 1.05–1.45 times higher degradation activity than ICCG in 4-d reaction at 50–70 °C, and pH 7.0 to pH 9.0, respectively. Furthermore, αSP-fusion lowered the optimal long-term degradation temperature from 65 °C to 50 °C at pH 8.0. Therefore, αSP-fusion was effective to increase the hydrolytic efficiency of PET hydrolases by increasing the degradation efficiency and decreasing the degradation temperature.

Quantitating α-amidated peptide degradation by separative technologies and ultra-high resolution mass spectrometry

α-amidation of peptides is a C-terminal modification allowing to improve half-life of therapeutic peptides. However, due to storage conditions, this C-terminal amide function can be partially hydrolyzed into a carboxylic acid moiety, resulting in a decrease of the molecule bioactivity. It is therefore necessary to be able to detect and quantify any trace of this degradation product when dealing with such class of peptides. For this purpose, LC-UV, CE-UV, LC/MS and LC/MS/MS are conventional analytical technologies used in pharmaceutical laboratories to monitor peptide integrity. Since the production of the carboxylated peptide issued from the amidated starting compound is scrutinized, ultra-high-resolution mass spectrometry was investigated without the recourse to any separative method (direct infusion mode) to achieve rapid straightforward relative quantitative analyses from the detected isotopic clusters of both peptides. Indeed, low or high-resolution MS instruments present in pharmaceutical laboratories (fitted with Q, IT or ToF mass analyzers) allow to distinguish both molecular ions that differ from 0.984 Da (NH2 formally substituted by OH). However, the analytical difficulty comes from the fact that the A+1 ion (13C contribution) of the amidated peptide and the A ion (12C contribution) of any produced carboxylated sequence are overlapping hampering the detection of minute amount of the hydrolyzed product. Actually, to differentiate these two isobaric ions, mass analyzers should possess at least a resolution of 90 000 for 7 residues peptides (molecular ions around 700–800 Da) which is presumably attainable with any FT-MS instrument. With an ultra-high-resolution mass spectrometer (FT-ICR MS) the observed fine isotopic structure displaying all expected isotopologues permitted in a single fast analysis to unambiguously detect both compounds (non-hydrolyzed and hydrolyzed one) and subsequently define the relative amount of the carboxylated peptide contaminant. Finally, these results were confronted with conventional measurements recorded with orthogonal methodologies such as LC-UV and CE-UV. The pro and cons of all quantitative strategies are discussed.

Effects of Charged Polyelectrolytes on Amyloid Fibril Formation of a Tau Fragment.

The microtubule-associated protein tau is involved in more than 20 different neurological disorders characterized by aberrant intracellular aggregation of tau in the brain. Here, we investigated the aggregation of a novel 20-residue model peptide, tau298-317, which is derived from the key microtubule binding domain of the full sequence tau. Our results show that tau298-317 highly mimics the physical and aggregation properties of tau. Under normal physiological conditions, the peptide maintains a disordered random coil without aggregation. The presence of polyanionic heparin (Hep) significantly promotes the aggregation of this peptide to form amyloid fibrils. The Hep-induced aggregation is sensitive to the ionic strength of the solution and the introduction of the negatively charged phosphate group on a serine (Ser305) residue in the sequence, suggesting an important role of electrostatic interactions in the mechanism of Hep-mediated aggregation. In addition, two positively charged polysaccharides, chitosan (CHT) and its quaternary derivative N-trimethyl chitosan (TMC), were found to effectively inhibit Hep-induced aggregation of tau298-317 in a concentration-dependent manner. Attractive electrostatic interactions between the positively charged moieties in CHT/TMC and the negatively charged residues of Hep play a critical role in inhibiting Hep-peptide interactions and suppressing peptide aggregation. Our results suggest that positively charged polyelectrolytes with optimized charged groups and charge distribution patterns can serve as effective molecular candidates to block tau-Hep interactions and prevent aggregation of tau induced by Hep and other polyanions.

Connecting sequence features within the disordered C-terminal linker of Bacillus subtilis FtsZ to functions and bacterial cell division

Intrinsically disordered regions (IDRs) can function as autoregulators of folded enzymes to which they are tethered. One example is the bacterial cell division protein FtsZ. This includes a folded core and a C-terminal tail (CTT) that encompasses a poorly conserved, disordered C-terminal linker (CTL) and a well-conserved 17-residue C-terminal peptide (CT17). Sites for GTPase activity of FtsZs are formed at the interface between GTP binding sites and T7 loops on cores of adjacent subunits within dimers. Here, we explore the basis of autoregulatory functions of the CTT in Bacillus subtilis FtsZ (Bs-FtsZ). Molecular simulations show that the CT17 of Bs-FtsZ makes statistically significant CTL-mediated contacts with the T7 loop. Statistical coupling analysis of more than 1,000 sequences from FtsZ orthologs reveals clear covariation of the T7 loop and the CT17 with most of the core domain, whereas the CTL is under independent selection. Despite this, we discover the conservation of nonrandom sequence patterns within CTLs across orthologs. To test how the nonrandom patterns of CTLs mediate CTT-core interactions and modulate FtsZ functionalities, we designed Bs-FtsZ variants by altering the patterning of oppositely charged residues within the CTL. Such alterations disrupt the core-CTT interactions, lead to anomalous assembly and inefficient GTP hydrolysis invitro and protein degradation, aberrant assembly, and disruption of cell division invivo. Our findings suggest that viable CTLs in FtsZs are likely to be IDRs that encompass nonrandom, functionally relevant sequence patterns that also preserve three-way covariation of the CT17, the T7 loop, and core domain.

Retrospective study for the universal applicability of the residue-based linear free energy relationship in the two-state exchange of protein molecules

Multiprobe measurements, such as NMR and hydrogen exchange studies, can provide the equilibrium constant, K, and rate constants for forward and backward processes, k and k′, of the two-state structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between log K and log k and between log K and log k′ for the topological exchange of a 27-residue bioactive peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific K, k, and k′ values in various exchange processes, including folding-unfolding equilibrium, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of the log–log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Among the successful cases, the hydrogen exchange study of apomyoglobin folding intermediates is particularly interesting. We found that the residues that deviated from the linear relationship corresponded to the α-helix, for which transient translocation had been identified by other experiments. Thus, the rbLFER is useful for studying the structures and energetics of the dynamic states of protein molecules.

Exploiting the Innate Plasticity of the Programmed Cell Death-1 (PD1) Receptor to Design Pembrolizumab H3 Loop Mimics.

Checkpoint blockade of the immunoreceptor programmed cell death-1 (PD1) with its ligand-1 (PDL1) by monoclonal antibodies such as pembrolizumab provided compelling clinical results in various cancer types, yet the molecular mechanism by which this drug blocks the PD1/PDL1 interface remains unclear. To address this question, we examined the conformational motion of PD1 associated with the binding of pembrolizumab. Our results revealed that the innate plasticity of both C'D and FG loops is crucial to form a deep binding groove (371 Å3 ) across several distant epitopes of PD1. This analysis ultimately provided a rational-design to create pembrolizumab H3 loop mimics [RDYRFDMGFD] into β-hairpin scaffolds. As a result, a 20-residue long β-hairpin peptide 1 e was identified as a first-in-class potent PD1-inhibitor (EC50 of 0.29 μM; Ki of 41 nM).

Design of Three Residues Peptides against SARS-CoV-2 Infection.

The continuous and rapid spread of the COVID-19 pandemic has emphasized the need to seek new therapeutic and prophylactic treatments. Peptide inhibitors are a valid alternative approach for the treatment of emerging viral infections, mainly due to their low toxicity and high efficiency. Recently, two small nucleotide signatures were identified in the genome of some members of the Coronaviridae family and many other human pathogens. In this study, we investigated whether the corresponding amino acid sequences of such nucleotide sequences could have effects on the viral infection of two representative human coronaviruses: HCoV-OC43 and SARS-CoV-2. Our results showed that the synthetic peptides analyzed inhibit the infection of both coronaviruses in a dose-dependent manner by binding the RBD of the Spike protein, as suggested by molecular docking and validated by biochemical studies. The peptides tested do not provide toxicity on cultured cells or human erythrocytes and are resistant to human serum proteases, indicating that they may be very promising antiviral peptides.

S3.4b Lactoferrin, a natural source of peptides that potentiate the antifungal activity of amphotericin B

S3.4 Free oral paper session, September 21, 2022, 4:45 PM - 6:15 PMObjectivesIt is notoriously difficult to prevent and treat fungal infections, however, the natural world has come up with remedies that are non-toxic, effective, and evade resistance. Here we investigate lactoferrin, an iron-binding glycoprotein found in milk, tears, and sweat, for its capacity to inhibit fungi and to synergize with commonly used antifungal drugs, with the aim of determining its mode of action.MethodsLactoferrin (LF) was obtained from a commercial supplier and two dairy companies. LF was tested on suite of pathogenic yeast and mold species for inhibition using CLSI microdilution methods. Synergy was determined with antifungal drugs amphotericin B (AMB), nystatin (NYS), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and 5-fluorocytosine (5FC). The effect of LF on fungal cells was analyzed using scanning electron microscopy (SEM). The active peptide/s within LF were then predicted from pepsin and in silico digestion, synthesized, and tested for synergy with amphotericin B (AMB). Tethered synthetic membranes were produced and were loaded with ergosterol or cholesterol to test the nature and specificity of membrane binding by LF and the synthetic peptide.ResultsLF demonstrated antifungal activity against yeast species Cryptococcus, Candida, and Saccharomyces and was much less effective against molds. Good synergy was achieved with AMB but not azole or echinocandin drugs. While the iron-chelating capacity of LF was important for the antifungal activity it was not involved in synergy. SEM revealed cell damage suggesting an interaction between AMB, LF and the fungal membrane or cell wall. A 30-residue peptide from the C lobe of LF was synthesized and tested for activity and synergy. This peptide, dubbed lactofungin (LFG) was inactive alone but was potently synergistic with AMB, indicating a direct role in augmenting AMB activity. Synthetic membranes loaded with ergosterol but not cholesterol were disrupted by AMB + LFG, demonstrating that activity was fungal-specific and was mediated through ergosterol binding.ConclusionLF is a complex molecule that causes fungal inhibition via iron binding and when cleaved by pepsin can produce active peptides. As AMB is a highly toxic treatment, the use of LFG as a synergent could help increase activity while lowering the effective dose, thereby reducing undesirable side effects. The action of AMB + LFG appears dependent on ergosterol, suggesting inhibition will be highly fungal-specific.

Discovery of a hidden form of neuropeptide F and its presence throughout the CNS–gut axis in the mud crab, Scylla olivacea

The mud crab Scylla olivacea (Scyol) is an economically crucial crustacean species in Thailand, due to its high market demand and nutritional value. The neuropeptide F (NPF) has been implicated in the coordinated regulation of feeding and metabolism in invertebrates. While various isoforms of neuropeptide F (NPF) have been previously explored in the mud crab, some knowledge gaps in relation to the NPF family, and ambiguities in the nomenclature from previous reports, remain. In this study, NPF was firstly localized in the central nervous system and gastrointestinal tract of the mud crab, S. olivacea, using a polyclonal antibody against Macrobrachium rosenbergii, Macro-NPF. The NPF immunoreactivity (ir) was detected dominantly in the X-organ/sinus gland complex of the eyestalk (ES) and the various neuronal clusters (cluster 6, 9/11, and 14/15) and neuropils (anteromedial and posteromedial protocerebral neuropils, olfactory and accessory olfactory neuropils, and medial antennule neuropil, columnar neuropil) of the brain (BR), commissural ganglia, and suboesophageal ganglion of the ventral nerve cord (VNC). Interestingly, this study also presented the NPF immunoreactivity (NPF-ir) in the acinar gland-like cell and spindle-shaped epithelial cells of S. olivacea intestine. The full-length Scyol-NPFII was characterized by molecular cloning and revealed 414 nucleotides with 375 nucleotides of an open reading frame which encoded 124 deduced amino acids. A 124-amino acid precursor protein of Scyol-NPFII included a 26-residue signal peptide and a 69-amino acid mature peptide. The Scyol-NPFII showed the highest percentage of hit similarity to S. paramamosain-NPFII and clustered in the NPFII family, separated from the other forms of NPFs in this species. The spatial gene expression in various tissues revealed that Scyol-NPFII was found dominantly in the ES and BR, VNC, heart, intestine, and muscle. This study provided a novel form of NPF in the female mud crab, S. olivacea, which could open the possibility of its functioning in the brain-to-gut controlling axis. This study could provide essential information for further application in the cultured system of S. olivacea in the near future.

Application study of infrared free-electron lasers towards the development of amyloidosis therapy.

Amyloidosis is known to be caused by the deposition of amyloid fibrils into various biological tissues; effective treatments for the disease are little established today. An infrared free-electron laser (IR-FEL) is an accelerator-based picosecond-pulse laser having tunable infrared wavelengths. In the current study, the irradiation effect of an IR-FEL was tested on an 11-residue peptide (NFLNCYVSGFH) fibril from β2-microglobulin (β2M) with the aim of applying IR-FELs to amyloidosis therapy. Infrared microspectroscopy (IRM) and scanning electron microscopy showed that a fibril of β2M peptide was clearly dissociated by IR-FEL at 6.1 µm (amide I) accompanied by a decrease of the β-sheet and an increase of the α-helix. No dissociative process was recognized at 6.5 µm (amide II) as well as at 5.0 µm (non-specific wavelength). Equilibrium molecular dynamics simulations indicated that the α-helix can exist stably and the probability of forming interchain hydrogen bonds associated with the internal asparagine residue (N4) is notably reduced compared with other amino acids after the β-sheet is dissociated by amide I specific irradiation. This result implies that N4 plays a key role for recombination of hydrogen bonds in the dissociation of the β2M fibril. In addition, the β-sheet was disrupted at temperatures higher than 340 K while the α-helix did not appear even though the fibril was heated up to 363 K as revealed by IRM. The current study gives solid evidence for the laser-mediated conversion from β-sheet to α-helix in amyloid fibrils at the molecular level.

Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate

Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel–selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel–lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.

A mutation in the S6 segment of the KvAP channel changes the secondary structure and alters ion channel activity in a lipid bilayer membrane.

The peptide segment S6 is known to form the inner lining of the voltage-gated K+ channel KvAP (potassium channel of archaea-bacterium, Aeropyrum pernix). In our previous work, it has been demonstrated that S6 itself can form an ion channel on a bilayer lipid membrane (BLM). In the present work, the role of a specific amino acid sequence 'LIG' in determining the secondary structure of S6 has been investigated. For this purpose, 22-residue synthetic peptides named S6-Wild (S6W) and S6-Mutant (S6M) were used. Sequences of these peptides are similar except that the two amino acids isoleucine and glycine of the wild peptide interchanged in the mutant peptide. Channel forming capabilities of both the peptides were checked electro-physiologically on BLM composed of DPhPC and cholesterol. Bilayer electrophysiological experiments showed that the conductance of S6M is higher than that of S6W. Significant differences in the current versus voltage (I-V) plot, open probability, and gating characteristics were observed. Interestingly, two sub-types of channels, S6M Type 1 and Type 2, were identified in S6M differing in conductances and open probability patterns. Circular dichroism (CD) spectroscopy indicated that the secondary structures of the two peptides are different in phosphatidyl choline/asolectin liposomes and 1% SDS detergent. Reduced helicity of S6M was also noticed in membrane mimetic liposomes and 1% SDS detergent micelles. These results are interpreted in view of the difference in hydrophobicity of the two amino acids, isoleucine and glycine. It is concluded that the 'LIG' stretch regulates the structure and pore-forming ability of the S6 peptide.

Humanin derivative, HNG, enhances neurotransmitter release

BackgroundHumanin (HN) is an endogenous 24-residue peptide that was first identified as a protective factor against neuronal death in Alzheimer's disease (AD). We previously demonstrated that the highly potent HN derivative HNG (HN with substitution of Gly for Ser14) ameliorated cognitive impairment in AD mouse models. Despite the accumulating evidence on the antagonizing effects of HN against cognitive deficits, the mechanisms behind these effects remain to be elucidated. MethodsThe extracellular fluid in the hippocampus of wild-type young mice was collected by microdialysis and the amounts of neurotransmitters were measured. The kinetic analysis of exocytosis was performed by amperometry using neuroendocrine cells. ResultsThe hippocampal acetylcholine (ACh) levels were increased by intraperitoneal injection of HNG. HNG did not affect the physical activities of the mice but modestly improved their object memory. In a neuronal cell model, rat pheochromocytoma PC12 cells, HNG enhanced ACh-induced dopamine release. HNG increased ACh-induced secretory events and vesicular quantal size in primary neuroendocrine cells. ConclusionsThese findings suggest that HN directly enhances regulated exocytosis in neurons, which can contribute to the improvement of cognitive functions. General significanceThe regulator of exocytosis is a novel physiological role of HN, which provides a molecular clue for HN's effects on brain functions under health and disease.

Design and Synthesis of certain Novel Peptides for Dual Selective Activity of Specific Cells

Cancer and the multi-resistant diseases that plague people today must be controlled to lower mortality rates. With the low susceptibility to resistance, the design and synthesis of peptide assembling was a superior alternative upgrading source for future chemotherapeutic medications. This paradigm first appeared in the pharmaceutical industry. It has been demonstrated that specific peptides exhibit dual functions as antibacterial and anticancer peptides (ACPs). Using a minimalized approach, a 10-residue peptide P1 and Rhodamine tagged peptide P2 were designed and synthesized using solid phase peptide synthesis method (SPPS) for selective activity against microbial and cancer cell lines. Peptide P1 contains RGD sequence has a net charge of +2 and peptide P2 has net charge of -4. The peptide P1 and P2 are characterized by using spectroscopic techniques. Circular dichroism studies showed changes in the secondary structure of peptide 1 and peptide 2 with buffer. Cytotoxicity assay exhibited the viability of normal and cancer cells up to 5 ?g concentrations of peptide 1.Thus, Peptide 1 acts on therapeutic properties like antibacterial and anticancer. Both peptide activities are checked against gram-positive and gram-negative bacteria at lower concentration. We can ensure the toxicity level of designed both peptides. Peptide P1 and peptide P2 sequence are non-toxic and recognition of selective activity against cancer cells.

Split luciferase-based estimation of cytosolic cargo concentration delivered intracellularly via attenuated cationic amphiphilic lytic peptides

Intracellular delivery of biomacromolecules is challenging as these molecules are taken up by cells and encapsulated into vesicular compartments called endosomes, and the fraction of molecules that are translocated to the cytosol are particularly important to obtain desired biological responses. This study aimed to estimate the cytosolic concentrations of intracellularly delivered peptides and proteins to aid the design of novel and effective biopharmaceutical delivery systems. To this end, we employed the split NanoLuc luciferase system, using the 11-residue HiBiT peptide segment as a probe for the delivered molecules in cells expressing the complementary LgBiT protein segment. The efficacy in cytosolic HiBiT delivery was determined by measuring the resultant luciferase activity when the HiBiT segment delivered into the cytosol forms a complex with LgBiT. Mean cytosolic HiBiT concentration was calculated using cell number and cell volume analysis. L17E and HAad peptides, developed in our laboratory for intracellular protein delivery, yielded approximately 6-fold cellular HiBiT concentrations than that obtained in their absence.

Computational Study of the Allosteric Effects of p5 on CDK5-p25 Hyperactivity as Alternative Inhibitory Mechanisms in Neurodegeneration.

The cyclin-dependent kinase (CDK5) forms a stable complex with its activator p25, leading to the hyperphosphorylation of tau proteins and to the formation of plaques and tangles that are considered to be one of the typical causes of Alzheimer's disease (AD). Hence, the pathological CDK5-p25 complex is a promising therapeutic target for AD. Small peptides, obtained from the truncation of CDK5 physiological activator p35, have shown promise in inhibiting the pathological complex effectively while also crossing the blood-brain barrier. One such small 24-residue peptide, p5, has shown selective inhibition toward the pathological complex in vivo. Our previous research focused on the characterization of a computationally predicted CDK5-p5 binding mode and of its pharmacophore, which was consistent with competitive inhibition. In continuation of our previous work, herein, we investigate four additional binding modes to explore other possible mechanisms of interaction between CDK5 and p5. The quantitative description of the pharmacophore is consistent with both competitive and allosteric p5-induced inhibition mechanisms of CDK5-p25 pathology. The gained insights can direct further in vivo/in vitro tests and help design small peptides, linear or cyclic, or peptidomimetic compounds as adjuvants of orthosteric inhibitors or as part of a cocktail of drugs with enhanced effectiveness and lower side effects.