Novel small synthetic HIV-1 V3 crown variants: CCR5 targeting ligands.

The CC chemokine receptor 5 (CCR5) antagonism represents a promising pharmacological strategy for therapeutic intervention as it plays a significant role in reducing the severity and progression of a wide range of pathological conditions. Here we designed and generated peptide ligands targeting the chemokine receptor, CCR5, that were derived from the critical interaction sites of the V3 crown domain of envelope protein glycoprotein gp120 (TRKSIHIGPGRAFYTTGEI) of HIV-1 using computational biology approach and the peptide sequence corresponding to this region was taken as the template peptide, designated as TMP-1. The peptide variants were synthesized by employing Fmoc chemistry using polymer support and were labelled with rhodamine B to study their interaction with the CCR5 receptor expressed on various cells. TMP-1 and TMP-2 were selected as the high-affinity ligands from in vitro receptor-binding assays. Specific receptor-binding experiments in activated peripheral blood mononuclear cells and HOS.CCR5 cells indicated that TMP-1 and TMP-2 had significant CCR5 specificity. Further, the functional analysis of TMP peptides using chemotactic migration assay showed that both peptides did not mediate the migration of responsive cells. Thus, template TMP-1 and TMP-2 represent promising CCR5 targeting peptide candidates.

Membrane Disruptive Action of Cationic Antibacterial Peptide B1CTcu3.

A twenty-two-residue peptide Brevinin1 Clinotarsus curtipus-3 (B1CTcu3), identified from the skin secretion of frog Clinotarsus curtipes of the Western Ghats, exhibited a broad range of antibacterial activity against Gram-negative and Gram-positive bacteria, including the methicillin-resistant Staphylococcus aureus (MRSA). It showed anti-biofilm activity even at sub-minimum inhibitory concentration (sub-MIC) against Pseudomonas aeruginosa and Staphylococcus aureus. Analysis of the scanning electron microscopic (SEM) images, confocal images, flow cytometric data and the effect of salt concentration on antibacterial potency suggests that the killing action of the peptide is through the membranolytic process. Single channel electric recording confirmed that the peptide elicited pores on the bacterial cell membrane as it induces a heterogeneous channel in the lipid bilayer. It also showed cytotoxicity against MDA-MB-231 breast cancer cell with IC50 of 25 µM. B1CTcu3 peptide could serve as the template for next-generation antibacterial agents, particularly against antibiotic resistant pathogenic bacteria.

Poly-Lysine Dendritic Nanocarrier to Target Epidermal Growth Factor Receptor Overexpressed Breast Cancer for Methotrexate Delivery.

A fourth generation poly-lysine dendritic nanocarrier (P4LDN)-based targeted chemotherapy for breast cancer is attempted by incorporating an epidermal growth factor receptor (EGFR)-specific short peptide E2 (ARSHVGYTGAR) and the drug methotrexate (MTX) into a nanocarrier system. The drug is incorporated into the nanocarrier using a cathepsin B cleavable spacer: glycine-phenylalanine-leucine-glycine (GFLG). The in vitro analysis of the time-dependent drug release, binding and internalization ability, and the cytotoxic nature showed that this drug delivery system (DDS) is highly effective. The efficacy analysis using non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice also showed that compared to the control group, the DDS can effectively reduce tumor volume. The mice that received the DDS appeared to gain weight more rapidly than the free drug, which suggests that the dendrimer is more easily tolerated by mice than the free drug.

Generation of hiPSC-derived low threshold mechanoreceptors containing axonal termini resembling bulbous sensory nerve endings and expressing Piezo1 and Piezo2.

Somatosensory low threshold mechanoreceptors (LTMRs) sense innocuous mechanical forces, largely through specialized axon termini termed sensory nerve endings, where the mechanotransduction process initiates upon activation of mechanotransducers. In humans, a subset of sensory nerve endings is enlarged, forming bulb-like expansions, termed bulbous nerve endings. There is no in vitro human model to study these neuronal endings. Piezo2 is the main mechanotransducer found in LTMRs. Recent evidence shows that Piezo1, the other mechanotransducer considered absent in dorsal root ganglia (DRG), is expressed at low level in somatosensory neurons. We established a differentiation protocol to generate, from iPSC-derived neuronal precursor cells, human LTMR recapitulating bulbous sensory nerve endings and heterogeneous expression of Piezo1 and Piezo2. The derived neurons express LTMR-specific genes, convert mechanical stimuli into electrical signals and have specialized axon termini that morphologically resemble bulbous nerve endings. Piezo2 is concentrated within these enlarged axon termini. Some derived neurons express low level Piezo1, and a subset co-express both channels. Thus, we generated a unique, iPSCs-derived human model that can be used to investigate the physiology of bulbous sensory nerve endings, and the role of Piezo1 and 2 during mechanosensation.

Maturational Changes in Mouse Cutaneous Touch and Piezo2-Mediated Mechanotransduction.

The age of studied animals has a profound impact on experimental outcomes in animal-based research. In mice, age influences molecular, morphological, physiological, and behavioral parameters, particularly during rapid postnatal growth and maturation until adulthood (at 12 weeks of age). Despite this knowledge, most biomedical studies use a wide-spanning age range from 4 to 12 weeks, raising concerns about reproducibility and potential masking of relevant age differences. Here, using mouse behavior and electrophysiology in cultured dorsal root ganglia (DRG), we reveal a decline in behavioral cutaneous touch sensitivity and Piezo2-mediated mechanotransduction in vitro during mouse maturation but not thereafter. In addition, we identify distinct transcript changes in individual Piezo2-expressing mechanosensitive DRG neurons by combining electrophysiology with single-cell RNA sequencing (patch-seq). Taken together, our study emphasizes the need for accurate age matching and uncovers hitherto unknown maturational plasticity in cutaneous touch at the level of behavior, mechanotransduction, and transcripts.

Myotubularin related protein-2 and its phospholipid substrate PIP2 control Piezo2-mediated mechanotransduction in peripheral sensory neurons.

Piezo2 ion channels are critical determinants of the sense of light touch in vertebrates. Yet, their regulation is only incompletely understood. We recently identified myotubularin related protein-2 (Mtmr2), a phosphoinositide (PI) phosphatase, in the native Piezo2 interactome of murine dorsal root ganglia (DRG). Here, we demonstrate that Mtmr2 attenuates Piezo2-mediated rapidly adapting mechanically activated (RA-MA) currents. Interestingly, heterologous Piezo1 and other known MA current subtypes in DRG appeared largely unaffected by Mtmr2. Experiments with catalytically inactive Mtmr2, pharmacological blockers of PI(3,5)P2 synthesis, and osmotic stress suggest that Mtmr2-dependent Piezo2 inhibition involves depletion of PI(3,5)P2. Further, we identified a PI(3,5)P2 binding region in Piezo2, but not Piezo1, that confers sensitivity to Mtmr2 as indicated by functional analysis of a domain-swapped Piezo2 mutant. Altogether, our results propose local PI(3,5)P2 modulation via Mtmr2 in the vicinity of Piezo2 as a novel mechanism to dynamically control Piezo2-dependent mechanotransduction in peripheral sensory neurons.

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons.

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.

Native Piezo2 Interactomics Identifies Pericentrin as a Novel Regulator of Piezo2 in Somatosensory Neurons.

The ability of somatosensory neurons to perceive mechanical stimuli relies on specialized mechanotransducing proteins and their molecular environment. Only recently has the identity of a major transducer of mechanical forces in vertebrates been revealed by the discovery of Piezo2. Further work has established its pivotal role for innocuous touch in mice. Therefore, Piezo2 offers a unique platform for the molecular investigation of somatosensory mechanosensation. We performed a mass spectrometry-based interactomics screen on native Piezo2 in somatosensory neurons of mouse dorsal root ganglia (DRG). Stringent and quantitative data analysis yielded the identity of 36 novel binding partners of Piezo2. The biological significance of this data set is reflected by functional experiments demonstrating a role for Pericentrin in modulating Piezo2 activity and membrane expression in somatosensory neurons. Collectively, our findings provide a framework for understanding Piezo2 physiology and serve as a rich resource for the molecular dissection of mouse somatosensation.

Alternatively Spliced Isoforms of KV10.1 Potassium Channels Modulate Channel Properties and Can Activate Cyclin-dependent Kinase in Xenopus Oocytes.

KV10.1 is a voltage-gated potassium channel expressed selectively in the mammalian brain but also aberrantly in cancer cells. In this study we identified short splice variants of KV10.1 resulting from exon-skipping events (E65 and E70) in human brain and cancer cell lines. The presence of the variants was confirmed by Northern blot and RNase protection assays. Both variants completely lacked the transmembrane domains of the channel and produced cytoplasmic proteins without channel function. In a reconstituted system, both variants co-precipitated with the full-length channel and induced a robust down-regulation of KV10.1 current when co-expressed with the full-length form, but their effect was mechanistically different. E65 required a tetramerization domain and induced a reduction in the overall expression of full-length KV10.1, whereas E70 mainly affected its glycosylation pattern. E65 triggered the activation of cyclin-dependent kinases in Xenopus laevis oocytes, suggesting a role in cell cycle control. Our observations highlight the relevance of noncanonical functions for the oncogenicity of KV10.1, which need to be considered when ion channels are targeted for cancer therapy.

Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief.

In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles.

Annexin A2 regulates TRPA1-dependent nociception.

The transient receptor potential A1 (TRPA1) channel is essential for vertebrate pain. Even though TRPA1 activation by ligands has been studied extensively, the molecular machinery regulating TRPA1 is only poorly understood. Using an unbiased proteomics-based approach we uncovered the physical association of Annexin A2 (AnxA2) with native TRPA1 in mouse sensory neurons. AnxA2 is enriched in a subpopulation of sensory neurons and coexpressed with TRPA1. Furthermore, we observe an increase of TRPA1 membrane levels in cultured sensory neurons from AnxA2-deficient mice. This is reflected by our calcium imaging experiments revealing higher responsiveness upon TRPA1 activation in AnxA2-deficient neurons. In vivo these findings are associated with enhanced nocifensive behaviors specifically in TRPA1-dependent paradigms of acute and inflammatory pain, while heat and mechanical sensitivity as well as TRPV1-mediated pain are preserved in AnxA2-deficient mice. Our results support a model whereby AnxA2 limits the availability of TRPA1 channels to regulate nociceptive signaling in vertebrates.