Signal Peptide Research Articles

Probing novel epitopes on the Plasmodium falciparum circumsporozoite protein for vaccine development

AbstractRTS,S and R21 are the only vaccines recommended by the WHO to protect children from Plasmodium falciparum (Pf) clinical malaria. Both vaccines target the Pf sporozoite surface protein circumsporozoite protein (CSP). Recent studies showed that human antibodies neutralize Pf sporozoites most efficiently when simultaneously binding to the PfCSP NANP repeat and the NPDP junction domain. However, neither RTS,S nor R21 targets this junction domain. To test the potential of the NPDP junction domain and other sites of PfCSP as innovative vaccine targets, we developed multiple vaccine candidates based on cucumber mosaic virus-like particles (CuMVTT-VLPs). These candidates vary in several aspects: the number of targeted NANP repeats, the presence or absence of the junction domain, the cleavage site, and up to three NVDP repeats within the target sequence. Immunogenicity and efficacy studies were conducted in BALB/c mice, utilizing chimeric Plasmodium berghei (Pb) sporozoites, in which the endogenous CSP has been replaced by PfCSP (Pb/PfCSP). We observed a positive association between the number of targeted NANP repeats and the induction of specific IgM/IgG antibodies. Elevated humoral responses led to enhanced protection against parasitemia after Pb/PfCSP sporozoite challenge. Especially high-avidity/affinity antibody formation and vaccine protection were NANP repeat-dependent. Intriguingly, vaccine efficacy was not enhanced by targeting sites on PfCSP other than the NANP repeats. Our data emphasize the dominant role of the NANP repeat region for induction of protective antibodies. Furthermore, we present here novel malaria vaccine candidates with an excellent immunogenic profile that confer sterile protection in mice, even in absence of adjuvants.

Acid Resistance Engineering of Endoglucanase for the Degradation of Wine Lees.

Wine lees is a low value biomass resource rich in cellulose, with great potential for producing organic fertilizers and chemicals. However, the high acidity of wine lees limits the catalytic efficiency of the conversion tool endoglucanase. Here, we expressed endoglucanase tCel5A from Trichoderma reesei in Pichia pastoris, and the combination of promoter AOX1 and signal peptide SUC2 resulted in a highly active expression of 4632.81 U/mg. Subsequently, the catalytic center design and surface charge modification strategy resulted in mutants T88H/W255H and S45D/T55D/T59D exhibiting catalytic activity twice and three times higher than WT at pH 3.0, respectively. Finally, when the solid-liquid ratio was 1:15 (w/v), the degradation rate of wine lees was nearly double that of WT. The degradation products contained a variety of industrial and pharmaceutical raw components, including the antioxidant and anticonvulsant isopiperolein B. This study accelerates the green and sustainable management of wine lees.

Identification of potential antigenic proteins and epitopes for the development of a monkeypox virus vaccine: an in silico approach.

Virus assembly, budding, or surface proteins play important roles such as viral attachment to cells, fusion, and entry into cells. The present study aimed to identify potential antigenic proteins and epitopes that could be used to develop a vaccine or diagnostic assay against the Monkeypox virus (MPXV) which may cause a potential epidemic. To do this, 39 MPXV proteins (including assembly, budding, and surface proteins) were analyzed using an in silico approach. Of these 39 proteins, the F5L virus protein was found to be the best vaccine candidate due to its signal peptide properties, negative GRAVY value, low transmembrane helix content, moderate aliphatic index, large molecular weight, long-estimated half-life, beta wrap motifs, and being stable, soluble, and containing non-allergic features. Moreover, the F5L protein exhibited alpha-helical secondary structures, making it a potential "structural antigen" recognized by antibodies. The other viral protein candidates were A9 and A43, but A9 lacked beta wrap motifs, while A43 had a positive GRAVY value and was insoluble. These two proteins were not as suitable candidates as the F5L protein. The KRVNISLTCL epitope from the F5L protein demonstrated the highest antigen score (2.4684) for MHC-I, while the GRFGYVPYVGYKCI epitope from the A9 protein exhibited the highest antigenicity (1.754) for MHC-II. Both epitopes met the criteria for high antigenicity, non-toxicity, solubility, non-allergenicity, and the presence of cleavage sites. Molecular docking and dynamics (MD) simulations further validated their potential, revealing stable and energetically favorable interactions with MHC molecules. The immunogenicity assessment showed that GRFGYVPYVGYKCI could strongly induce immune responses through both IFN-γ and IL-4 pathways, suggesting its capacity to provoke a balanced Th1 and Th2 response. In contrast, KRVNISLTCL exhibited limited immunostimulatory potential. Overall, these findings lay the groundwork for future vaccine development, indicating that F5L, particularly the GRFGYVPYVGYKCI epitope, may serve as an effective candidate for peptide-based vaccine design against MPXV.

Engineering Saccharomyces cerevisiae for continuous secretory production of hEGF in biofilm.

Human epidermal growth factor (hEGF) plays a crucial role in promoting cell growth and has various clinical applications. Due to limited natural sources and the high cost of chemical synthesis, researchers are now exploring genetic engineering as a potential method for hEGF production. In this particular study, a novel hEGF expression system was developed using Saccharomyces cerevisiae. This system involved optimizing the promoter and signal peptide and deleting protease-coding genes PEP4, PRB1, and YAP3, overexpressing chaperones KAR2 and PDI1 in the protein secretion pathway, which led to a 2.01-fold increase in hEGF production compared to the wild type strain. Furthermore, biofilm-forming genes FLO11 and ALS3 were integrated to create a biofilm strain with adhesive properties. A biofilm-based immobilized continuous fermentation model was established to leverage the characteristics of this biofilm strain. Each batch of this model yielded 130mg/L of hEGF, with a production efficiency of 2.71mg/L/h - surpassing the production efficiency of traditional free fermentation (1.62mg/L/h). This study presents a promising fermentation model for efficient hEGF production based on biofilm characteristics, offering valuable insights for the application of biofilm fermentation in the production of small molecule peptides.

DeepmiRNATar: A deep learning-based model for miRNA targets prediction

MicroRNAs (miRNAs) play a crucial role in regulating fundamental biological processes such as the cell cycle, differentiation, and apoptosis by directly interacting with multiple genes (mRNAs). This regulatory mechanism has a profound impact on cellular function and the overall physiological condition of an organism. However, the prediction of miRNA-mRNA interactions encounters computational challenges in the field of biology due to the diverse sequences and complex data patterns. To overcome these obstacles, this research effort introduced DeepmiRNATar, a tool designed to precisely pinpoint miRNA targets, offering essential assistance in the realm of disease management. DeepmiRNATar leverages the Word2vec-based DeepLncLoc approach for encoding miRNA sequence characteristics and utilizes the DNABERT pre-trained model for in-depth semantic comprehension of target sequences. Through the integration of TextCNN, BiLSTM, and SpatialConv Attention mechanisms, the model scrutinizes structural features, temporal relationships, and overall interactions within the sequences. Following a series of experimental assessments, DeepmiRNATar attained an impressive AUC of 99.15% on the evaluation dataset, on par with the current leading prediction methodologies. Notably, the precision-recall curve, sensitivity, and F-measure values reached 99.18%, 97.43%, and 95.47%, respectively. Compared to existing miRNA target prediction models, DeepmiRNATar demonstrates a notable enhancement in overall predictive accuracy. The successful creation and experimental validation of the DeepmiRNATar model signify a significant advancement in miRNA target identification technology.

Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae.

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1' position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities.

Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein.

Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be modified to bind to a specific ssRNA nucleobase. As such, there is interest in developing 'designer' PPRs (dPPRs) for a range of biotechnology applications, including diagnostics or in vivo localization of ssRNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. To address this, we determined the structure of a dPPR bound to its target sequence and used two- and three-color single-molecule fluorescence resonance energy transfer to interrogate the mechanism of ssRNA binding to individual dPPRs in real time. We demonstrate that dPPRs are slower to bind longer ssRNA sequences (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR. Importantly, dPPR binds only to its target sequence (i.e. it does not associate with non-target ssRNA sequences) and does not 'scan' longer ssRNA oligonucleotides for the target sequence. The kinetic constraints imposed by random 3D diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus).

NcPTP2, a polar tube protein, interacts with spore wall protein in the parasitic microsporidian Nosema ceranae.

Microsporidia is an obligate intracellular eukaryote, which is capable of parasitizing vertebrates and invertebrates. Nosema ceranae, which can infect both Apis mellifera and Apis cerana, poses a serious threat and causes heavy losses to the worldwide apiculture. During infection, polar tube, a highly specialized invasion structure, ejected from the spore to deliver the sporoplasm into host cells to cause infection. Although seven different polar tube proteins (PTP1 ~ 7) have been reported from various microsporidia and showed key functions associated with spore invasion and proliferation, no systematic analysis on identification and characterization of polar tube proteins from N. ceranae was found. The polar tube proteins 2 (NcPTP2) was identified from the total polar tube proteins of N. ceranae by LC_MS/MS and the transcriptional profile was performed by RT-PCR. Sequence characterization analysis revealed that NcPTP2 was rich in lysine and had a signal peptide at the N-terminal. It had 3 potential O-glycosylation sites and 6 potential N-glycosylation sites. 25 phosphorylation sites were found on serine, tyrosine and threonine sites. Sequence alignment analysis revealed that NcPTP2 was homologous and had conserved cysteine residues with PTP2 proteins from other microsporidia. Indirect immunofuorescence analysis (IFA) and Immunoelectron Microscopy analysis (IEM) confirmed that NcPTP2 was localized on the polar tube of the germinated spores. The interaction between NcPTP2 and spore wall protein in N. ceranae indicated its potential function in anchoring and coiling of polar tube in spore. NcPTP2 was the first subcellular localized polar tube protein in N. ceranae and this work could provide an important basis for further analyzing the biological functions of polar tube proteins and uncovering the infection mechanism of N. ceranae to the host cells.

Abstract 4137688: Inhibition of Cardiac Fibrosis and Pro-Fibrotic Collagen Hormone Endotrophin by VS-041, a Novel Drug Candidate for Heart Failure with Preserved Ejection Fraction

Introduction: Endotrophin, a collagen type VI–derived signaling peptide, has been linked to cardiac metabolic dysregulation, inflammation, and fibrosis. Several matrix metalloproteinases (MMPs), incl. MMP2 and MMP9, have been shown to cleave collagen 6A3 and release endotrophin. Baseline plasma endotrophin levels in heart failure with preserved ejection fraction (HFpEF) patients were shown to be strongly and independently associated with increased risk of poor outcomes (death or HF-admissions). VS-041 is a novel drug candidate for HFpEF that inhibits key MMPs implicated in cardiac fibrosis and diastolic dysfunction. Previously, VS-041 demonstrated robust efficacy in the Dahl Salt Sensitive rat model of hypertension and HFpEF, improving diastolic function and dose-proportionally reducing left ventricle (LV) fibrosis. Hypothesis: Inhibition of endotrophin release by VS-041 could contribute to its anti-fibrotic mechanism and serve as a potential biomarker of MMP inhibition (target engagement) in patients. Methods and Results: The effect of VS-041 on production of endotrophin was investigated in a “Scar-in-a-Jar” model using primary human cardiac fibroblasts isolated from adult healthy ventricles. Fibroblasts were treated with 10 ng/mL platelet-derived growth factor (PDGF) AB in the presence of 0.03, 0.3, or 3 µM VS-041, or DMSO control. The concentration of endotrophin in the medium was assessed via the ELISA nordicPRO-C6 TM (Nordic Bioscience A/S). After 4 days of treatment, VS-041 at 0.3 µM and 3 µM significantly inhibited PDGF AB-stimulated production of endotrophin. Cumulative inhibitory effects of VS-041 could still be observed after 12 days of culture. The antifibrotic activity of VS-041 was also tested in a model of extensive cardiac fibrosis in 16-months old C57BL/6 mice exacerbated by angiotensin II infusion at 1 mg/kg/day for 28 days. Oral treatment with VS-041 at 75 mg/kg BID resulted in a 62% reduction (p<0.01) of LV fibrosis as assessed by morphometry of Masson trichrome stained sections. VS-041 also improved the diastolic echocardiography parameters IVRT and E/a ratio. Conclusions: The production of endotrophin by fibroblasts is diminished by VS-041 and, therefore, its plasma levels could be explored as a biomarker of in vivo MMP inhibition by VS-041. Additionally, the inhibition of endotrophin release by VS-041 could be causatively linked to reduction of cardiac fibrosis and potentially translate into improved clinical outcomes in HFpEF patients.

Abstract 4131674: ADP-Ribosylation In a Mouse Model of Atherosclerosis: a Potential Novel Link Between Dyslipidemia and Inflammation in Cardiovascular Disease

Background: Inflammation and lipid accumulation are major features of atherosclerosis, a leading cause of death and morbidity worldwide. Our previous study recognized ADP-ribosylation, a post-translational modification, as a novel regulator of macrophage activation. We also have established mass spectrometry-based ADP-ribosylation proteomics. Using this technology, we evaluated the completely uncharacterized role of ADP-ribosylation in atherogenesis. We hypothesized that ADP-ribosylated proteins circulate from liver, accumulate in aorta and promote atherogenesis. Methods and Results: We harvested the aorta, liver, and plasma of LDL receptor-deficient ( Ldlr -/- ) mice that were on a regular chow or high-fat diet for 3 or 6 months (n=40/condition). To increase ADP-ribosyl peptide signals in the aorta, we applied our novel recently optimized ion mobility mass spectrometry strategy to generate ADP-ribosylation proteomics data. We analyzed 160 mice aortas and identified 3 APOA1 and 3 APOE ADP-ribosylated peptides in both the aorta and the liver (Fig. A). In addition, these peptides were differentially abundant in the aorta of HFD-fed mice, compared to controls (i.e, APOA1 ARPALEDLR peptide relative abundance (Fig. B)). Using the same mouse plasma, we then validated the presence of ADP-ribosylated APOA1 and ADP-ribosylated APOE in HDL and chylomicron/VLDL/LDL fractions (Western blot), respectively. This finding indicates that classical apolipoproteins circulate as ADP-ribosylated forms, representing a completely novel class of modified apolipoproteins. Immunohistochemistry confirmed the enrichment of aortic lesions in macrophages and ADP-ribosylation signal (5-fold increase, p=0.0006). Conclusions: This work provides the first in vivo evidence that ADP-ribosylation occurs in atherosclerotic lesions, which may originate from the liver via circulating blood (Fig. C). Future studies will examine whether ADP-ribosylation of apolipoproteins, specifically APOA1, alters anti-atherogenic functions of HDL.

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization.

Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.

DDDR-10. MYELOID CELLS COORDINATELY INDUCE GLIOMA CELL-INTRINSIC AND -EXTRINSIC PATHWAYS FOR CHEMORESISTANCE VIA GP130 SIGNALING

Abstract The DNA damage response (DDR) and the blood tumor barrier (BTB) restrict chemotherapeutic success for primary brain tumors like glioblastoma (GBM). Coherently, GBM almost invariably relapse with fatal outcome. We here show that interaction of GBM and myeloid cells simultaneously induces chemoresistance on the genetic and the vascular level by activating GP130 receptor signaling, which can be addressed therapeutically. We performed transcriptomic and immunohistochemical screens with human brain material, pharmacological experiments with a humanized organotypic GBM model, proteomics and cell-based assays and observed that nanomolar concentrations of the signaling peptide Humanin promoted TMZ resistance through DDR activation. GBM mouse models recapitulating intratumoral Humanin-release showed accelerated BTB formation. Genetic analysis of pericyte to endothelial cell signaling in transgenic mice revealed increased GP130 activation in Humanin releasing tumors as compared to controls. GP130 blockade attenuated both DDR activity and BTB formation resulting in improved preclinical chemotherapeutic efficacy. Altogether, we describe an overarching mechanism for TMZ resistance and outline a translatable strategy with predictive markers to improve chemotherapy for GBM.

A molecular toolkit for heterologous protein secretion across Bacteroides species.

Bacteroides species are abundant, prevalent, and stable members of the human gut microbiota, making them a promising chassis for developing long-term interventions for chronic diseases. Engineering Bacteroides as in situ bio-factories, however, requires efficient protein secretion tools, which are currently lacking. Here, we systematically investigate methods to enable heterologous protein secretion in Bacteroides. We identify a collection of secretion carriers that can export functional proteins across multiple Bacteroides species at high titers. To understand the mechanistic drivers of Bacteroides secretion, we characterize signal peptide sequence features, post-secretion extracellular fate, and the size limit of protein cargo. To increase titers and enable flexible control of protein secretion, we develop a strong, self-contained, inducible expression circuit. Finally, we validate the functionality of our secretion carriers in vivo in a mouse model. This toolkit promises to enable expanded development of long-term living therapeutic interventions for chronic gastrointestinal disease.

Clinical Applications of Antisense Oligonucleotides in Cancer: A Focus on Glioblastoma

Antisense oligonucleotides (ASOs) are promising drugs capable of modulating the protein expression of virtually any target with high specificity and high affinity through complementary base pairing. However, this requires a deep understanding of the target sequence and significant effort in designing the correct complementary drug. In addition, ASOs have been demonstrated to be well tolerated during their clinical use. Indeed, they are already used in many diseases due to pathogenic RNAs of known sequences and in several neurodegenerative diseases and metabolic diseases, for which they were given marketing authorizations (MAs) in Europe and the United States. Their use in oncology is gaining momentum with several identified targets, promising preclinical and clinical results, and recent market authorizations in the US. However, many challenges remain for their clinical use in cancer. It seems necessary to take a step back and review our knowledge of ASOs and their therapeutic uses in oncology. The objectives of this review are (i) to summarize the current state of the art of ASOs; (ii) to discuss the therapeutic use of ASOs in cancer; and (iii) to focus on ASO usage in glioblastoma, the challenges, and the perspective ahead.

CNSC-36. REGULATION OF CELL FATE IN PAEDIATRIC HIGH-GRADE GLIOMA BY THE HOMEOBOX TRANSCRIPTION FACTOR DLX2

Abstract Paediatric high-grade gliomas (pHGG) are nearly uniformly fatal cancers that arise predominantly in children and adolescents and encompass Diffuse Midline Glioma (DMG) and Diffuse Hemispheric Glioma (DHG). Most patients harbour mutations in histone variants H3.1 or H3.3, resulting in global epigenetic dysregulation and a stalled oligodendroglial progenitor (OPC) or GABAergic interneuron progenitor (IP) fate for DMG and DHG, respectively. DLX2, a homeobox transcription factor, represses OPC fate and promotes IP cell fate and migration during early neurogenesis. Our lab has shown that Dlx2 binds to and regulates the promoters of Gad1/Gad2, as well as early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of Dlx2 with these target sequences reduces reporter gene expression in vitro and Dlx1/2 double knockout mice exhibit increased expression of these OPC markers in vivo. Transient overexpression of a Dlx2-GFP construct in murine DMG cells led to significant IP marker increase (Gad1/2), decrease of OPC markers (Olig2, Nkx2.2) and global restoration of H3K27me3. Additionally, decreased proliferation, colony growth, invasion and migration were observed in vitro. To translate these findings in human pHGG, analysis of a bulk RNA-seq database of 62 patient-derived pHGG cell lines revealed an inverse relationship between DLX2 and OLIG2. DLX2 was knocked out of DLX2high/OLIG2low DHG cell lines using CRISPR-Cas9, and overexpressed in DLX2low/OLIG2high DMG cell lines using a DLX2-mCherry overexpression construct. Evaluation of cell fate markers and epigenetic marks will be presented. Changes in migration, invasion, and proliferation in vitro will also be presented, in addition to changes in tumour growth using a neonatal xenograft murine model in vivo. Manipulation of DLX2 in pHGG provides a model for investigating the link between the factors that control cell fate decisions in the developing brain and the aberrant differentiation state in pHGG.

Structure and Function of Sabinene Synthase, a Monoterpene Cyclase That Generates a Highly Strained [3.1.0] Bicyclic Product.

Sabinene is a plant natural product with a distinctive strained [3.1.0] bicyclic ring system that is used commercially as a spicy and pine-like fragrance with citrus undertones. This unusual monoterpene has also been studied as an antifungal and anti-inflammatory agent as well as a next-generation biofuel. In order to understand the molecular determinants of [3.1.0] bicyclic ring formation in sabinene biosynthesis, we now report three X-ray crystal structures of sabinene synthase from Western red cedar, Thuja plicata (TpSS), with open and partially closed active site conformations at 2.21-2.72 Å resolution. We additionally report the complete biochemical characterization of sabinene synthase, including steady-state kinetics, active site mutagenesis, and product array profiling. The catalytic metal ion requirement is unexpectedly broad for a class I terpene cyclase: optimal catalytic activity was measured using Mn2+ or Co2+, with more modest activity observed using Mg2+ or Ni2+. Kinetic parameters were determined for both full-length TpSS and a deletion variant lacking the putative N-terminal plastidial targeting sequence, designated ΔTpSS. Monoterpene product profiles for both indicated similar product arrays independent of the catalytic metal ion used, with sabinene comprising nearly 90% of the total products generated. Site-directed mutagenesis was utilized to probe the function of active site residues, and several mutants yielded altered product arrays. Most notably, the G458A substitution converted ΔTpSS into a high-activity α-pinene synthase. α-Pinene contains a bicyclic [3.1.1] ring system; structural and mechanistic analyses suggest a molecular rationale for the reprogrammed transannulation reaction, leading to the alternative bicyclic product.

Peptidergic G-Protein-Coupled Receptor Signaling Systems in Cancer: Examination of Receptor Structure and Signaling to Foster Innovative Pharmacological Solutions

Background. Peptidergic GPCR systems are broadly distributed in the human body and regulate numerous physiological processes by activating complex networks of intracellular biochemical events responsible for cell regulation and survival. Excessive stimulation, ill-function, or blockade of GPCRs produces cell disturbances that may cause disease should compensatory mechanisms not suffice. Methods and Results. Revision of updated experimental research provided an evident relationship associating peptidergic GPCR malfunction with tumor formation and maintenance resulting from uncontrolled cell proliferation and migration, colonization, inhibition of apoptosis or altered metabolism, and increased angiogenesis in tumoral tissues. Conclusion. Determination of the implication of GPCR peptide signaling in specific neoplasia is crucial to designing tailored pharmacological treatments to counteract or dismantle the origin of the signaling circuitry causing cellular disruption. In some cases, particular ligands for these receptors may serve as concomitant treatments to aid other pharmacological or physical approaches to eradicate neoplasias.

Effect of biphasic in vitro maturation (CAPA-IVM) on EGF receptor and embryo development of prepubertal goat oocytes according to follicle size

Oocytes spontaneously resume meiosis following their liberation from follicles, preventing full competence acquisition. Biphasic IVM (CAPA-IVM) maintains oocytes in meiotic arrest to improve developmental competence, and it specially affects poorly developed oocytes. We assessed the effect of CAPA-IVM on oocytes from small (<3mm) and large (>3mm) follicles of prepubertal goats. Oocytes were cultured for 6h in pre-IVM with C-type natriuretic peptide (CNP) and estradiol as meiotic inhibitors, and germinal vesicle (GV) rate and chromatin configuration were assessed. Then, oocytes were cultured in conventional IVM (c-IVM) or CAPA-IVM (pre-IVM + c-IVM) and EGF receptor (EGFR) protein expression, intra-oocyte ROS and blastocyst development were assessed. GV rate was higher in CNP groups than control (69% vs 28%, and 67% vs 31%, small and large follicles, respectively), but GV chromatin configuration was similar. In large follicles, EGFR expression was higher in oocytes and cumulus cells after CAPA-IVM, and ROS levels were lower. In small follicles these differences were not observed. c-IVM and CAPA-IVM produced similar blastocyst rates in small (3.7% vs 2.6%, respectively) and large follicles (8.3% vs 2.5%). Overall, CAPA-IVM enhanced EGFR expression for EGF peptide signalling and antioxidant capacity in oocytes from large follicles but oocytes from small follicles were too immature to benefit from it.

Crowdsourcing Adverse Events Associated With Monoclonal Antibodies Targeting Calcitonin Gene-Related Peptide Signaling for Migraine Prevention: Natural Language Processing Analysis of Social Media.

Clinical trials demonstrate the efficacy and tolerability of medications targeting calcitonin gene-related peptide (CGRP) signaling for migraine prevention. However, these trials may not accurately reflect the real-world experiences of more diverse and heterogeneous patient populations, who often have higher disease burden and more comorbidities. Therefore, postmarketing safety surveillance is warranted. Regulatory organizations encourage marketing authorization holders to screen digital media for suspected adverse reactions, applying the same requirements as for spontaneous reports. Real-world data from social media platforms constitute a potential venue to capture diverse patient experiences and help detect treatment-related adverse events. However, while social media holds promise for this purpose, its use in pharmacovigilance is still in its early stages. Computational linguistics, which involves the automatic manipulation and quantitative analysis of oral or written language, offers a potential method for exploring this content. This study aims to characterize adverse events related to monoclonal antibodies targeting CGRP signaling on Reddit, a large online social media forum, by using computational linguistics. We examined differences in word frequencies from medication-related posts on the Reddit subforum r/Migraine over a 10-year period (2010-2020) using computational linguistics. The study had 2 phases: a validation phase and an application phase. In the validation phase, we compared posts about propranolol and topiramate, as well as posts about each medication against randomly selected posts, to identify known and expected adverse events. In the application phase, we analyzed posts discussing 2 monoclonal antibodies targeting CGRP signaling-erenumab and fremanezumab-to identify potential adverse events for these medications. From 22,467 Reddit r/Migraine posts, we extracted 402 (2%) propranolol posts, 1423 (6.33%) topiramate posts, 468 (2.08%) erenumab posts, and 73 (0.32%) fremanezumab posts. Comparing topiramate against propranolol identified several expected adverse events, for example, "appetite," "weight," "taste," "foggy," "forgetful," and "dizziness." Comparing erenumab against a random selection of terms identified "constipation" as a recurring keyword. Comparing erenumab against fremanezumab identified "constipation," "depression," "vomiting," and "muscle" as keywords. No adverse events were identified for fremanezumab. The validation phase of our study accurately identified common adverse events for oral migraine preventive medications. For example, typical adverse events such as "appetite" and "dizziness" were mentioned in posts about topiramate. When we applied this methodology to monoclonal antibodies targeting CGRP or its receptor-fremanezumab and erenumab, respectively-we found no definite adverse events for fremanezumab. However, notable flagged words for erenumab included "constipation," "depression," and "vomiting." In conclusion, computational linguistics applied to social media may help identify potential adverse events for novel therapeutics. While social media data show promise for pharmacovigilance, further work is needed to improve its reliability and usability.

Topology surveillance of the lanosterol demethylase CYP51A1 by Signal Peptide Peptidase.

Cleavage of transmembrane segments on target proteins by the aspartyl intramembrane protease signal peptide peptidase (SPP) has been linked to immunity, viral infection and protein quality control. How SPP recognizes its various substrates and specifies their fate remains elusive. Here we identified the lanosterol demethylase CYP51A1 as an SPP substrate and show that SPP-catalyzed cleavage triggers CYP51A1 clearance by ER-associated degradation (ERAD). We observe that SPP targets only a fraction of CYP51A1 molecules and identified an amphipathic helix in the N-terminus as a key determinant for SPP recognition. SPP recognition is remarkably specific to CYP51A1 molecules with the amphipathic helix aberrantly inserted in the membrane with a type II orientation. Thus, our data are consistent with a role for SPP in topology surveillance, triggering the clearance of certain, potentially non-functional conformers.