Optimal regulation of finite Markov Chains

Imperial Users onl

The cationic tetradecapeptide mastoparan as a privileged structure for drug discovery: Enhanced antimicrobial properties of mitoparan analogues modified at position-14

Mastoparan (MP) peptides, distributed in insect venoms, induce a local inflammatory response post envenomation. Most endogenous MPs share common structural elements within a tetradecapeptide sequence that adopts an amphipathic helix whilst traversing biological membranes and when bound to an intracellular protein target. Rational modifications to increase cationic charge density and amphipathic helicity engineered mitoparan (MitP), a mitochondriotoxic bioportide and potent secretagogue. Following intracellular translocation, MitP is accreted by mitochondria thus indicating additional utility as an antimicrobial agent. Hence, the objectives of this study were to compare the antimicrobial activities of a structurally diverse set of cationic cell penetrating peptides, including both MP and MitP sequences, and to chemically engineer analogues of MitP for potential therapeutic applications. Herein, we confirm that, like MP, MitP is a privileged structure for the development of antimicrobial peptides active against both prokaryotic and eukaryotic pathogens. Collectively, MitP and target-selective chimeric analogues are broad spectrum antibiotics, with the Gram-negative A. baumannii demonstrating particular susceptibility. Modifications of MitP by amino acid substitution at position-14 produced peptides, Δ14MitP analogues, with unique pharmacodynamic properties. One example, [Ser14]MitP, lacks both cytotoxicity against human cell lines and mast cell secretory activity yet retains selective activity against the encapsulated yeast C. neoformans

Mitoparans: mitochondriotoxic cell penetrating peptides and novel inducers of apoptosis.

Acknowledgments The authors would like to thank Keith Holding at the University of Wolverhampton for his outstanding technical support. This work was supported in part by Samantha Dickson Brain Tumour Trust.Introduction: The amphipathic helical peptide mastoparan (MP; H-INLKALAALAKKIL-NH2) inserts into biological membranes to modulate the activity of heterotrimeric G proteins and other targets. Moreover, whilst cell free models of apoptosis demonstrate MP to facilitate mitochondrial permeability transition and release of apoptogenic cytochrome c, MP-induced death of intact cells has been attributed to its non-specific membrane destabilising properties (necrotic mechanisms). However, MP and related peptides are known to activate other signalling systems, including p42/p44 MAP kinases and could therefore, also modulate cell fate and specific apoptotic events. The ability of MP to facilitate mitochondrial permeability in cell free systems has lead to proposals that MP could be of utility in tumour therapeutics provided that it conferred features of cellular penetration and mitochondrial localization. We have recently reported that our highly potent amphipathic MP analogue mitoparan (mitP; [Lys5,8Aib10]MP; Aib = -aminoisobutyric acid) specifically promotes apoptosis of human cancer cells, as was confirmed by in situ TUNEL staining and activation of caspase-3. Moreover, we have also demonstrated that mitP penetrates plasma membranes and redistributes to co-localize with mitochondria. Complementary studies, using isolated mitochondria, further demonstrated that mitP, through co-operation with a protein of the permeability transition pore complex voltage-dependent anion channel (VDAC), induced swelling and permeabilization of mitochondria, leading to the release of the apoptogenic factor cytochrome c. An expanding field of peptide and cell penetrating peptide (CPP) research has focussed on the selective targeting of tumours by engineering constructs that incorporate cell-specific or tissue–specific address motifs. Peptidyl address motifs could enhance the selectivity of drug delivery whilst the improved cellular uptake offered by CPP enhances bioavailability. Thus and as a potential therapeutic strategy, we extended our findings to design target-specific mitP analogues. The integrin-specific address motif RGD and a Fas ligand mimetic WEWT were incorporated by N-terminal acylation of mitP to produce novel tandem-linked chimeric peptides

Exploring the effect of exercise training on testicular function

Purpose The impact of exercise training on testicular function is relatively ill-defined. To gain new insights into this important topic, published data, deriving from both humans and animal studies, were critically analyzed. Results and conclusions The effects of exercise on the hypothalamus–pituitary–gonadal axis, influenced by the type, intensity and duration of the exercise program, can be evaluated in terms of total and free testosterone and/or luteinizing hormone and follicle-stimulating hormone serum levels and sperm parameters. High-intensity exercise promotes a common decrease in these parameters, and therefore, negatively impacts upon testicular function. However, published data for moderate-intensity exercise training are inconsistent. Conversely, there is consistent evidence to support the benefits of exercise training to prevent and/or counteract the impairment of testis function caused by aging, obesity and doxorubicin treatment. This positive effect is likely the consequence of decreased oxidative stress and inflammatory status. In the future, it will be important to clarify the molecular mechanisms which explain these reported discrepancies and to establish guidelines for an active lifestyle to promote healthy testicular function.publishe

A new biology of cell penetrating peptides

This is an accepted manuscript of an article published by Wiley in Peptide Science on 05/03/2020, available online: https://doi.org/10.1002/pep2.24154 The accepted version of the publication may differ from the final published version.As pharmacokinetic modifiers, cell penetrating peptides (CPPs) have proven utility for the delivery of otherwise impermeable cargoes into the discrete intracellular compartments of eukaryotic cells. Methods for the molecular optimisation of CPP sequences can significantly increase the performance of novel vectors matched to a specific delivery function. Moreover, the ‘information rich’ composition and inherent molecular flexibility of many CPPs can also facilitate their interaction with intracellular proteins and other cellular structures. This proteomimetic property of CPPs has been exploited in the design of bioportides, bioactive CPPs that regulate cellular functions often by binding relatively flat PPI interfaces to achieve a dominant negative action. This new biology of CPPs is rapidly gathering momentum through the design and synthesis of a bewildering variety of peptides that may be, in whole or part, linear, helical, cyclic and/or chimeric in nature. A particular emphasis of contemporary CPP‐centred drug discovery is the unmet medical need of cancer, though the biomedical scope of bioportide applications is impressively broad including cellular signalling and reprogramming. We are hopeful that one or more of the fascinating studies described herein will translate into the clinic to establish bioportide technologies as a viable option for drug discovery.British Council; Fundação para a Ciência e a Tecnologia; Michael J. Fox Foundation for Parkinson's Research.Published onlin

Cell-penetrating peptides, targeting the regulation of store-operated channels, slow decay of the progesterone-induced [Ca 2+ ] i signal in human sperm

© 2015 The Authors. Published by Oxford University Press. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1093/molehr/gav019Previous work has provided evidence for involvement of store-operated channels (SOCs) in [Ca(2+)]i signalling of human sperm, including a contribution to the transient [Ca(2+)]i elevation that occurs upon activation of CatSper, a sperm-specific cation channel localized to the flagellum, by progesterone. To further investigate the potential involvement of SOCs in the generation of [Ca(2+)]i signals in human sperm, we have used cell-penetrating peptides containing the important basic sequence KIKKK, part of the STIM-Orai activating region/CRAC activating domain (SOAR/CAD) of the regulatory protein stromal interaction molecule 1. SOAR/CAD plays a key role in controlling the opening of SOCs, which occurs upon mobilization of stored Ca(2+). Resting [Ca(2+)]i temporarily decreased upon application of KIKKK peptide (3-4 min), but scrambled KIKKK peptide had a similar effect, indicating that this action was not sequence-specific. However, in cells pretreated with KIKKK, the transient [Ca(2+)]i elevation induced by stimulation with progesterone decayed significantly more slowly than in parallel controls and in cells pretreated with scrambled KIKKK peptide. Examination of single-cell responses showed that this effect was due, at least in part, to an increase in the proportion of cells in which the initial transient was maintained for an extended period, lasting up to 10 min in a subpopulation of cells. We hypothesize that SOCs contribute to the progesterone-induced [Ca(2+)]i transient, and that interference with the regulatory mechanisms of SOC delays their closure, causing a prolongation of the [Ca(2+)]i transient.L.L. was supported by theWellcome Trust (Grant #086470). J.M. was supported by a University of Birmingham Teaching Assistantship. Funding to pay the Open Access publication charges for this article was provided by . .

A high-throughput synthetic platform enables the discovery of proteomimetic cell penetrating peptides and bioportides

Collectively, cell penetrating peptide (CPP) vectors and intrinsically active bioportides possess tremendous potential for drug delivery applications and the discrete modulation of intracellular targets including the sites of protein–protein interactions (PPIs). Such sequences are usually relatively short (< 25 AA), polycationic in nature and able to access the various intracellular compartments of eukaryotic cells without detrimental influences upon cellular biology. The high-throughput platform for bioportide discovery described herein exploits the discovery that many human proteins are an abundant source of potential CPP sequences which are reliably predicted using QSAR algorithms or other methods. Subsequently, microwave-enhanced solid phase peptides synthesis provides a high-throughput source of novel proteomimetic CPPs for screening purposes. By focussing upon cationic helical domains, often located within the molecular interfaces that facilitate PPIs, bioportides which act by a dominant-negative mechanism at such sites can be reliably identified within small number libraries of CPPs. Protocols that employ fluorescent peptides, routinely prepared by N-terminal acylation with carboxytetramethylrhodamine, further enable both the quantification of cellular uptake kinetics and the identification of specific site(s) of intracellular accretion. Chemical modifications of linear peptides, including strategies to promote and stabilise helicity, are compatible with the synthesis of second-generation bioportides with improved drug-like properties to further exploit the inherent selectivity of biologics

Stem cell bioengineering with bioportides: inhibition of planarian head regeneration with peptide mimetics of eyes absent proteins

© 2023 The Authors. Published by MDPI AG. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3390/pharmaceutics15082018Djeya1 (RKLAFRYRRIKELYNSYR) is a very effective cell penetrating peptide (CPP) that mimics the α5 helix of the highly conserved Eya domain (ED) of eyes absent (Eya) proteins. The objective of this study was to bioengineer analogues of Djeya1 that, following effective translocation into planarian tissues, would reduce the ability of neoblasts (totipotent stem cells) and their progeny to regenerate the anterior pole in decapitated S. mediterranea. As a strategy to increase the propensity for helix formation, molecular bioengineering of Djeya1 was achieved by the mono-substitution of the helicogenic aminoisobutyric acid (Aib) at three species-variable sites: 10, 13, and 16. CD analyses indicated that Djeya1 is highly helical, and that Aib-substitution had subtle influences upon the secondary structures of bioengineered analogues. Aib-substituted Djeya1 analogues are highly efficient CPPs, devoid of influence upon cell viability or proliferation. All three peptides increase the migration of PC-3 cells, a prostate cancer line that expresses high concentrations of Eya. Two peptides, [Aib13]Djeya1 and [Aib16]Djeya1, are bioportides which delay planarian head regeneration. As neoblasts are the only cell population capable of division in planaria, these data indicate that bioportide technologies could be utilised to directly manipulate other stem cells in situ, thus negating any requirement for genetic manipulation.Bárbara Matos was supported by an individual grant from the Portuguese Foundation for Science and Technology (FCT), grant identifier SFRH/BD/146032/2019.Published onlin

Disruption of Protein Phosphatase 1 complexes using bioportides as a novel approach to target sperm motility

This is an accepted manuscript of an article published by Elsevier in Fertility and Sterility on 23/09/2020, available online: https://doi.org/10.1016/j.fertnstert.2020.08.013 The accepted version of the publication may differ from the final published version.To design protein phosphatase 1 (PP1)-disrupting peptides covalently coupled to inert cell penetrating peptides (CPPs) as sychnologically-organized bioportide constructs as a strategy to modulate sperm motility. Design: Experimental study. Setting: Academic research laboratory. Patients/Animals: Normozoospermic men providing samples for routine analysis and Holstein Frisian bulls. Intervention(s): None. Main Outcome Measure(s): Effect of the bioportides on the activity and interactions of PP1γ2 – a PP1 isoform expressed exclusively in testicular germ cells and sperm - and on sperm vitality and motility. Results: PP1‐disrupting peptides were designed based on the sequences from (i) a sperm-specific PP1 interactor (A kinase anchor protein 4, AKAP4) and (ii) a PP1 inhibitor (protein phosphatase inhibitor 2, PPP1R2). Those sequences were covalently coupled to inert CPPs as bioportide constructs, which were successfully delivered to the flagellum of sperm cells to induce a marked impact upon PP1γ2 activity and sperm motility. Molecular modelling studies further facilitated the identification of an optimized PP1 binding sequence and enabled the development of a Modified Stop Sperm (MSS1) bioportide with reduced size and increased potency of action. Additionally, a bioportide mimetic of the unique 22-amino acid C-terminus of PP1γ2 accumulated within spermatozoa to significantly reduce sperm motility and further define the PP1γ2-specific interactome. Conclusion: These investigations demonstrate the utility of CPPs to deliver peptide sequences 53 that target unique protein-protein interactions in spermatozoa to achieve a significant impact upon 54 spermatozoa motility, a key prognostic indicator of male fertility.This work was supported by FEDER funds through the “Programa Operacional Competitividade e Internacionalização COMPETE 2020” and by National Funds through the FCT Fundação para a Ciência e Tecnologia (PTDB/BBB-BQB/3804/2014). We are thankful to iBiMED (UIDB/04501/2020 and POCI-01-0145-FEDER-007628) for supporting this project. This work was also supported by an individual grant from FCT to J.V.S. (SFRH/BPD/123155/2016)

Chronic exercise training attenuates prostate cancer-induced molecular remodelling in the testis

This is an accepted manuscript of an article published by Springer in Cellular Oncology 0n 19/10/2020, available online at: https://doi.org/10.1007/s13402-020-00567-9 The accepted version of the publication may differ from the final published version.Purpose Prostate cancer is a major cause of cancer-related death in males worldwide and, in addition to impairing prostate function, also causes testicular adaptations. In this study, we aim to investigate the preventive effect of exercise training on PCa-induced testicular dysfunction. Methods As a model, we used fifty Wistar Unilever male rats, randomly divided in four experimental groups. Prostate cancer was chemically and hormonally induced in two groups of animals (PCa groups). One control group and one PCa group was submitted to moderate intensity treadmill exercise training. Fifty weeks after the start of the training the animals were sacrificed and sperm, prostate, testes and serum were collected and analyzed. Sperm concentration and morphology, and testosterone serum levels were determined. In addition, histological analysis of the testes was performed, and testis proteomes and metabolomes were characterized. Results We found that prostate cancer negatively affected testicular function, manifested as an arrest of spermatogenesis. Oxidative stress-induced DNA damage, arising from reduced testis blood flow, may also contribute to apoptosis of germ cells and consequential spermatogenic impairment. Decreased utilization of the glycolytic pathway, increased metabolism of ketone bodies and the accumulation of branched chain amino acids were also evident in the PCa animals. Conversely, we found that the treadmill training regimen activated DNA repair mechanisms and counteracted several metabolic alterations caused by PCa without impact on oxidative stress. Conclusions These findings confirm a negative impact of prostate cancer on testis function and suggest a beneficial role for exercise training in the prevention of prostate cancer-induced testis dysfunction