Class Of Proteins Research Articles

New promising routes in peptic ulcers: Toll-like receptors and semaphorins.

Peptic ulcers (PU) are one of the commonest yet problematic diseases found to be existing in the majority of the population. Today, drugs from a wide range of therapeutic classes are available for the management of the disease. Still, the complications of the condition are difficult to tackle and the side effect profile is quite a concern. The literature indicates that Toll-like receptors (TLRs) and Semaphorins (SEMAs) have been under study for their various pharmacological actions over the past few decades. Both these signalling pathways are found to regulate immunological and inflammatory responses. Moreover, receptors and signalling molecules from the family of TLRs and SEMAs are found to have bacterial recognition and antibacterial properties which are essential in eradicating Helicobacter pylori (H. pylori), one of the major causative agents of PU. Our understanding of SEMAs, a class of proteins involved in cell signalling, is relatively less developed compared to TLRs, another class of proteins involved in the immune response. SEMAs and TLRs play different roles in biological processes, with SEMAs primarily involved in guiding cell migration and axon guidance during development, while TLRs are responsible for recognizing pathogens and initiating an immune response. Here, in this review, we will discuss in detail the signalling cascade of TLRs and SEMAs and thereby understand its association with PU for future therapeutic targeting. The review also aims at providing an overview of the study that has been into exploring the role of these signalling pathways in the management of PU.

Structure classification of the proteins from Salmonella enterica pangenome revealed novel potential pathogenicity islands

Salmonella enterica is a pathogenic bacterium known for causing severe typhoid fever in humans, making it important to study due to its potential health risks and significant impact on public health. This study provides evolutionary classification of proteins from Salmonella enterica pangenome. We classified 17,238 domains from 13,147 proteins from 79,758 Salmonella enterica strains and studied in detail domains of 272 proteins from 14 characterized Salmonella pathogenicity islands (SPIs). Among SPIs-related proteins, 90 proteins function in the secretion machinery. 41% domains of SPI proteins have no previous sequence annotation. By comparing clinical and environmental isolates, we identified 3682 proteins that are overrepresented in clinical group that we consider as potentially pathogenic. Among domains of potentially pathogenic proteins only 50% domains were annotated by sequence methods previously. Moreover, 36% (1330 out of 3682) of potentially pathogenic proteins cannot be classified into Evolutionary Classification of Protein Domains database (ECOD). Among classified domains of potentially pathogenic proteins the most populated homology groups include helix-turn-helix (HTH), Immunoglobulin-related, and P-loop domains-related. Functional analysis revealed overrepresentation of these protein in biological processes related to viral entry into host cell, antibiotic biosynthesis, DNA metabolism and conformation change, and underrepresentation in translational processes. Analysis of the potentially pathogenic proteins indicates that they form 119 clusters or novel potential pathogenicity islands (NPPIs) within the Salmonella genome, suggesting their potential contribution to the bacterium’s virulence. One of the NPPIs revealed significant overrepresentation of potentially pathogenic proteins. Overall, our analysis revealed that identified potentially pathogenic proteins are poorly studied.

Interpretable molecular encodings and representations for machine learning tasks

Molecular encodings and their usage in machine learning models have demonstrated significant breakthroughs in biomedical applications, particularly in the classification of peptides and proteins. To this end, we propose a new encoding method: Interpretable Carbon-based Array of Neighborhoods (iCAN). Designed to address machine learning models' need for more structured and less flexible input, it captures the neighborhoods of carbon atoms in a counting array and improves the utility of the resulting encodings for machine learning models. The iCAN method provides interpretable molecular encodings and representations, enabling the comparison of molecular neighborhoods, identification of repeating patterns, and visualization of relevance heat maps for a given data set. When reproducing a large biomedical peptide classification study, it outperforms its predecessor encoding. When extended to proteins, it outperforms a lead structure-based encoding on 71% of the data sets. Our method offers interpretable encodings that can be applied to all organic molecules, including exotic amino acids, cyclic peptides, and larger proteins, making it highly versatile across various domains and data sets. This work establishes a promising new direction for machine learning in peptide and protein classification in biomedicine and healthcare, potentially accelerating advances in drug discovery and disease diagnosis.

Epigenetics in etiopathology of hyperprolactinemia

Aim: Epigenetic alterations have been reported in patients with pituitary tumors and those on antipsychotic drugs, which are also responsible for hyperprolactinemia. This suggests a possible role of epigenetics in the etiopathology of hyperprolactinemia. Methods: The study recruited 83 hyperprolactinemia cases with prolactin > 100 ng/mL and 65 controls. Global DNA methylation status was studied by MethylFlash Methylated DNA Quantification Kit and genome-wide methylation analysis (GWMA) by Infinium Methylation EPIC BeadChip 850K array. Results: Hyperprolactinemia cases showed significant global DNA hypermethylation compared to controls. Around 66.67% of hypomethylated and 12.9% of hypermethylated cases were on antipsychotics. Gene enrichment analysis of 5-cytosine-phosphate-guanine-3 (CpG) site-associated genes demonstrated significantly enriched major histocompatibility complex (MHC)-related protein classes and cellular components. Conclusions: The study suggested the role of epigenetics in the etiopathology of hyperprolactinemia.

Angiomotin family proteins in the Hippo signaling pathway.

The Motin family proteins (Motins) are a class of scaffolding proteins consisting of Angiomotin (AMOT), AMOT-like protein 1 (AMOTL1), and AMOT-like protein 2 (AMOTL2). Motins play a pivotal role in angiogenesis, tumorigenesis, and neurogenesis by modulating multiple cellular signaling pathways. Recent findings indicate that Motins are components of the Hippo pathway, a signaling cascade involved in development and cancer. This review discusses how Motins are integrated into the Hippo signaling network, as either upstream regulators or downstream effectors, to modulate cell proliferation and migration. The repression of YAP/TAZ by Motins contributes to growth inhibition, whereas subcellular localization of Motins and their interactions with actin fibers are critical in regulating cell migration. The net effect of Motins on cell proliferation and migration may contribute to their diverse biological functions.

Involvement of Chemosensory Protein CrufCSP3 in Perception of the Host Location in a Parasitic Wasp Cotesia ruficrus.

Chemosensory proteins (CSPs) constitute a class of olfactory proteins localized in insect sensory organs that serve a crucial function in decoding external chemical stimuli. This study aims to elucidate the involvement of CrufCSP3 in olfactory perception within the context of Cotesia ruficrus, an indigenous endoparasitoid targeting the invasive pest Spodoptera frugiperda. Through fluorescence-competitive binding assays and site-directed mutagenesis, we pinpointed four amino acids as pivotal residues involved in the interaction between CrufCSP3 and five host-related compounds. Subsequent RNA interference experiments targeting CrufCSP3 unveiled a reduced sensitivity to specific host-related compounds and a decline in the parasitism rate of the FAW larvae. These findings unequivocally indicate the essential role of CrufCSP3 in the chemoreception process of C. ruficrus. Consequently, our study not only sheds light on the functional importance of CSPs in parasitic wasp behavior but also contributes to the development of eco-friendly and efficacious wasp behavior modifiers for effectively mitigating pest population surges.

Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no β-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Amantadine interactions with phase separated lipid membranes

Amantadine, a small amphilphic organic compound that consists of an adamantane backbone and an amino group, was first recognized as an antiviral in 1963 and received approval for prophylaxis against the type A influenza virus in 1976. Since then, it has also been used to treat Parkinson’s disease-related dyskinesia and is being considered as a treatment for corona viruses. Since amantadine usually targets membrane-bound proteins, its interactions with the membrane are also thought to be important. Biological membranes are now widely understood to be laterally heterogeneous and certain proteins are known to preferentially co-localize within specific lipid domains. Does amantadine, therefore, preferentially localize in certain lipid composition domains? To address this question, we studied amantadine’s interactions with phase separating membranes composed of cholesterol, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), as well as single-phase DPhPC (1,2-diphytanoyl-sn-glycero-3-phos-phocholine) membranes. From Langmuir trough and differential scanning calorimetry (DSC) measurements, we determined, respectively, that amantadine preferentially binds to disordered lipids, such as POPC, and lowers the phase transition temperature of POPC/DSPC/cholesterol mixtures, implying that amantadine increases membrane disorder. Further, using droplet interface bilayers (DIBs), we observed that amantadine disrupts DPhPC membranes, consistent with its disordering properties. Finally, we carried out molecular dynamics (MD) simulations on POPC/DSPC/cholesterol membranes with varying amounts of amantadine. Consistent with experiment, MD simulations showed that amantadine prefers to associate with disordered POPC-rich domains, domain boundaries, and lipid glycerol backbones. Since different proteins co-localize with different lipid domains, our results have possible implications as to which classes of proteins may be better targets for amantadine.

Label free quantitative proteomic profiling of serum samples of intellectually disabled young patients revealed dysregulation of complement coagulation and cholesterol cascade systems.

Intellectual disability is a heterogeneous disorder, diagnosed using intelligence quotient (IQ) score criteria. Currently, no specific clinical test is available to diagnose the disease and its subgroups due to inadequate understanding of the pathophysiology. Therefore, current study was designed to explore the molecular mechanisms involved in disease perturbation, and to identify potential biomarkers for disease diagnosis and prognosis. A total of 250 participants were enrolled in this study, including 200 intellectually disabled (ID) subjects from the subgroups (mild, moderate, and severe) with age and gender matched healthy controls (n = 50). Initially, IQ testing score and biochemical profile of each subject was generated, followed by label-free quantitative proteomics of subgroups of IQ and healthy control group through nano-LC/MS- mass spectrometry. A total of 310 proteins were identified, among them198 proteins were common among all groups. Statistical analysis (ANOVA) of the subgroups of ID showed 142 differentially expressed proteins, in comparison to healthy control group. From these, 120 proteins were found to be common among all subgroups. The remaining 22 proteins were categorized as exclusive proteins found only in disease subgroups. Furthermore, the hierarchical cluster analysis (HCL) of common significant proteins was also performed, followed by PANTHER protein classification and GO functional enrichment analysis. Results provides that the datasets of differentially expressed proteins, belong to the categories of immune / defense proteins, transfer carrier proteins, apolipoproteins, complement proteins, protease inhibitors, hemoglobin proteins etc., they are known to involvein immune system, and complement and coagulation pathway cascade and cholesterol metabolism pathway. Exclusively expressed 22 proteins were found to be disease stage specific and strong PPI network specifically those that have significant role in platelets activation and degranulation, such as Filamin A (FLNA). Furthermore, to validate the mass spectrometric findings, four highly significant proteins (APOA4, SAP, FLNA, and SERPING) were quantified by ELISA in all the study subjects. AUROC analysis showed a significant association of APOA4 (0.830), FLNA (0.958), SAP (0.754) and SERPING (0.600) with the disease. Apolipoprotein A4 (APOA4) has a significant role in cholesterol transport, and in modulation of glucose and lipid metabolism in the CNS. Similarly, FLNA has a crucial role in the nervous system, especially in the functioning of synaptic network. Therefore, both APOA4, and FLNA proteins represent good potential for candidate biomarkers for the diagnosis and prognosis of the intellectual disability. Overall, serum proteome of ID patients provides valuable information of proteins/pathways that are altered during ID progression.

Deciphering the function of the fifth class of Gα proteins: regulation of ionic homeostasis as unifying hypothesis

Trimeric G proteins transduce signals from a superfamily of receptors and each G protein controls a wide range of cellular and systemic functions. Their highly conserved alpha subunits fall in five classes, four of which have been well investigated (Gs, Gi, G12, Gq). In contrast, the function of the fifth class, Gv is completely unknown, despite its broad occurrence and evolutionary ancient origin (older than metazoans). Here we show a dynamic presence of Gv mRNA in several organs during early development of zebrafish, including the hatching gland, the pronephros and several cartilage anlagen, employing in situ hybridisation. Next, we generated a Gv frameshift mutation in zebrafish and observed distinct phenotypes such as reduced oviposition, premature hatching and craniofacial abnormalities in bone and cartilage of larval zebrafish. These phenotypes could suggest a disturbance in ionic homeostasis as a common denominator. Indeed, we find reduced levels of calcium, magnesium and potassium in the larvae and changes in expression levels of the sodium potassium pump atp1a1a.5 and the sodium/calcium exchanger ncx1b in larvae and in the adult kidney, a major osmoregulatory organ. Additionally, expression of sodium chloride cotransporter slc12a3 and the anion exchanger slc26a4 is altered in complementary ways in adult kidney. It appears that Gv may modulate ionic homeostasis in zebrafish during development and in adults. Our results constitute the first insight into the function of the fifth class of G alpha proteins.

The association of protein-bound methionine sulfoxide with proteomic basis for aging in beech seeds

BackgroundEuropean beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared.ResultsAged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds.ConclusionsWe demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.

The Shigella kinase effector OspG modulates host ubiquitin signaling to escape septin-cage entrapment

Shigella flexneri is a Gram-negative bacterium causing severe bloody dysentery. Its pathogenesis is largely dictated by a plasmid-encoded type III secretion system (T3SS) and its associated effectors. Among these, the effector OspG has been shown to bind to the ubiquitin conjugation machinery (E2~Ub) to activate its kinase activity. However, the cellular targets of OspG remain elusive despite years of extensive efforts. Here we show by unbiased phosphoproteomics that a major target of OspG is CAND1, a regulatory protein controlling the assembly of cullin-RING ubiquitin ligases (CRLs). CAND1 phosphorylation weakens its interaction with cullins, which is expected to impact a large panel of CRL E3s. Indeed, global ubiquitome profiling reveals marked changes in the ubiquitination landscape when OspG is introduced. Notably, OspG promotes ubiquitination of a class of cytoskeletal proteins called septins, thereby inhibiting formation of cage-like structures encircling cytosolic bacteria. Overall, we demonstrate that pathogens have evolved an elaborate strategy to modulate host ubiquitin signaling to evade septin-cage entrapment.

Molecular evolution of Phytocyanin gene and analysis of expression at different coloring periods in apple (Malus domestica)

BackgroundPC (phytocyanin) is a class of copper-containing electron transfer proteins closely related to plant photosynthesis, abiotic stress responses growth and development in plants, and regulation of the expression of some flavonoids and phenylpropanoids, etc., however, compared with other plants, the PC gene family has not been systematically characterized in apple.ResultsA total of 59 MdPC gene members unevenly distributed across 12 chromosomes were identified at the genome-wide level. The proteins of the MdPC family were classified into four subfamilies based on differences in copper binding sites and glycosylation sites: Apple Early nodulin-like proteins (MdENODLs), Apple Uclacyanin-like proteins (MdUCLs), Apple Stellacyanin-like proteins (MdSCLs), and Apple Plantacyanin-like proteins (MdPLCLs). Some MdPC members with similar gene structures and conserved motifs belong to the same group or subfamily. The internal collinearity analysis revealed 14 collinearity gene pairs among members of the apple MdPC gene. Interspecific collinearity analysis showed that apple had 31 and 35 homologous gene pairs with strawberry and grape, respectively. Selection pressure analysis indicated that the MdPC gene was under purifying selection. Prediction of protein interactions showed that MdPC family members interacted strongly with the Nad3 protein. GO annotation results indicated that the MdPC gene also regulated the biosynthesis of phenylpropanoids. Chip data analysis showed that (MdSCL3, MdSCL7 and MdENODL27) were highly expressed in mature fruits and peels. Many cis-regulatory elements related to light response, phytohormones, abiotic stresses and flavonoid biosynthetic genes regulation were identified 2000 bp upstream of the promoter of the MdPC gene, and qRT-PCR results showed that gene members in Group IV (MdSCL1/3, MdENODL27) were up-regulated at all five stages of apple coloring, but the highest expression was observed at the DAF13 (day after fruit bag removal) stage. The gene members in Group II (MdUCL9, MdPLCL3) showed down-regulated or lower expression in the first four stages of apple coloring but up-regulated and highest expression in the DAF 21 stage.ConclusionHerein, one objective of these findings is to provide valuable information for understanding the structure, molecular evolution, and expression pattern of the MdPC gene, another major objective in this study was designed to lay the groundwork for further research on the molecular mechanism of PC gene regulation of apple fruit coloration.

Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogen Phyllachora maydis.

Phyllachora maydis is an ascomycete foliar fungal pathogen and the causal agent of tar spot in maize. Though P. maydis is considered an economically important foliar pathogens of maize, our general knowledge of the trophic lifestyle and functional role of effector proteins from this fungal pathogen remains limited. Here, we utilized a genome-informed approach to predict the trophic lifestyle of P. maydis and functionally characterized a subset of candidate effectors from this fungal pathogen. Leveraging the most recent P. maydis genome annotation and the CATAStrophy pipeline, we show this fungal pathogen encodes a predicted Carbohydrate-active enzymes (CAZymes) repertoire consistent with that of biotrophs. To investigate fungal pathogenicity, we selected 18 candidate effector proteins that were previously shown to be expressed during primary disease development. We assessed whether these putative effectors share predicted structural similarity with other characterized fungal effectors and determined whether any suppress plant immune responses. Using AlphaFold2 and Foldseek, we showed one candidate effector, PM02_g1115, adopts a predicted protein structure similar to that of an effector from Verticillium dahlia. Furthermore, transient expression of candidate effector-fluorescent protein fusions in Nicotiana benthamiana revealed two putative effectors, PM02_g378 and PM02_g2610, accumulated predominantly in the cytosol, and three candidate effectors, PM02_g1115, PM02_g7882, and PM02_g8240 consistently attenuated chitin-mediated reactive oxygen species production. Collectively, these results presented herein provide insights into the predicted trophic lifestyle and putative functions of effectors from P. maydis and will likely stimulate continued research to elucidate the molecular mechanisms used by P. maydis to induce tar spot.

FgPfn participates in vegetative growth, sexual reproduction, pathogenicity, and fungicides sensitivity via affecting both microtubules and actin in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB), caused by Fusarium graminearum species complexes (FGSG), is an epidemic disease in wheat and poses a serious threat to wheat production and security worldwide. Profilins are a class of actin-binding proteins that participate in actin depolymerization. However, the roles of profilins in plant fungal pathogens remain largely unexplored. Here, we identified FgPfn, a homolog to profilins in F. graminearum, and the deletion of FgPfn resulted in severe defects in mycelial growth, conidia production, and pathogenicity, accompanied by marked disruptions in toxisomes formation and deoxynivalenol (DON) transport, while sexual development was aborted. Additionally, FgPfn interacted with Fgα1 and Fgβ2, the significant components of microtubules. The organization of microtubules in the ΔFgPfn was strongly inhibited under the treatment of 0.4 μg/mL carbendazim, a well-known group of tubulin interferers, resulting in increased sensitivity to carbendazim. Moreover, FgPfn interacted with both myosin-5 (FgMyo5) and actin (FgAct), the targets of the fungicide phenamacril, and these interactions were reduced after phenamacril treatment. The deletion of FgPfn disrupted the normal organization of FgMyo5 and FgAct cytoskeleton, weakened the interaction between FgMyo5 and FgAct, and resulting in increased sensitivity to phenamacril. The core region of the interaction between FgPfn and FgAct was investigated, revealing that the integrity of both proteins was necessary for their interaction. Furthermore, mutations in R72, R77, R86, G91, I101, A112, G113, and D124 caused the non-interaction between FgPfn and FgAct. The R86K, I101E, and D124E mutants in FgPfn resulted in severe defects in actin organization, development, and pathogenicity. Taken together, this study revealed the role of FgPfn-dependent cytoskeleton in development, DON production and transport, fungicides sensitivity in F. graminearum.

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-28-12.

Proteomic comparison of the organic matrices from parietal and base plates of the acorn barnacle Amphibalanus amphitrite.

Acorn barnacles are efficient colonizers on a wide variety of marine surfaces. As they proliferate on critical infrastructure, their settlement and growth have deleterious effects on performance. To address acorn barnacle biofouling, research has focused on the settlement and adhesion processes with the goal of informing the development of novel coatings. This effort has resulted in the discovery and characterization of several proteins found at the adhesive substrate interface, i.e. cement proteins, and a deepened understanding of the function and composition of the biomaterials within this region. While the adhesive properties at the interface are affected by the interaction between the proteins, substrate and mechanics of the calcified base plate, little attention has been given to the interaction between the proteins and the cuticular material present at the substrate interface. Here, the proteome of the organic matrix isolated from the base plate of the acorn barnacle Amphibalanus amphitrite is compared with the chitinous and proteinaceous matrix embedded within A. amphitrite parietal plates. The objective was to gain an understanding of how the basal organic matrix may be specialized for adhesion via an in-depth comparative proteome analysis. In general, the majority of proteins identified in the parietal matrix were also found in the basal organic matrix, including nearly all those grouped in classes of cement proteins, enzymes and pheromones. However, the parietal organic matrix was enriched with cuticle-associated proteins, of which ca 30% of those identified were unique to the parietal region. In contrast, ca 30-40% of the protease inhibitors, enzymes and pheromones identified in the basal organic matrix were unique to this region. Not unexpectedly, nearly 50% of the cement proteins identified in the basal region were significantly distinct from those found in the parietal region. The wider variety of identified proteins in the basal organic matrix indicates a greater diversity of biological function in the vicinity of the substrate interface where several processes related to adhesion, cuticle formation and expansion of the base synchronize to play a key role in organism survival.

Signaling of GPR17 in Hepatic and Renal Tissues

G protein-coupled receptors (GPCRs) are the largest class of proteins in the human body and are considered to be excellent targets for drug discovery. However, the vast majority of GPCRs are understudied. Using a large-scale TaqMan array screen on both male and female C57BL6 murine livers, we identified GPR17 as one of the most highly expressed orphan receptors within the liver (ΔCt = 19.9, normalized to GAPDH). GPR17 expression in the liver was confirmed using reverse transcriptase polymerase chain reaction (RT-PCR) and was also identified in murine whole kidney from both males and females indicating that GPR17 RNA expression is not sex-dependent. While GPR17 has known roles in oligodendrocyte maturation and myelination in the central nervous system, its functions within the liver and kidney are undetermined. To investigate its physiological functions, we started by uncovering its localization within the liver and kidney and identifying its ligand profile. A series of RNAScope in-situ hybridization assays were performed to localize GPR17 within hepatic tissue. Gpr17 mRNA was found within hepatocytes indicating that its highest expression is in the parenchymal cells of the liver. To identify possible activators for GPR17, we cloned the coding region into both flag-tagged PME18s and the Presto-Tango vectors. Live-cell surface labeling of HEK293T expressing the GPR17 constructs confirmed robust surface expression on the plasma membrane facilitating ligand screening. GPR17 downstream signaling has yet to be studied. Thus, we used the PRESTO-TANGO assay, an exogenous chemical ligand screening assay that relies on a luminescence readout to indicate -arrestin recruitment and receptor activation. A large, non-biased chemical screen of over 500 physiological compounds was tested for activation of GPR17. To date, we identified seven exogenous activators of GPR17: 3-[methyl(1,2,3,4- tetrahydronaphthalen-2-yl)amino]pyrazine-2-carbonitrile, 4-(4-fluorophenyl)-5-methyl-N-(2-methyl-1-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)thiophene-2-carboxamide,({1-[(2-chlorophenyl)methyl]-3,5-dimethyl-1H-pyrazol-4-yl}methyl)(methyl)[(1-methyl-1H-imidazol-2-yl)methyl]amine,N,N-dimethyl-6-[(5-phenyl-1,3-oxazol-2-yl)methyl]-5H,6H,7H,8H-pyrido[4,3-d]pyrimidin-4-amine,4-phenyl-1-[6-(pyrrolidin-1-yl)-9H-purin-9-yl]butan-2-ol, 2-(3-fluorophenyl)-3-methyl-1-{[1,2,4]triazolo[4,3-a]pyridine-6-carbonyl}pyrrolidine, and 2,6,6-trimethyl-N-({2-methyl-4H,5H,6H,7H-pyrazolo[1,5-a]pyrimidin-6-yl}methyl)-4,5,6,7-tetrahydro-1-benzofuran-4-amine. Ongoing studies are aimed at narrowing down the in vivo activators of GPR17 to ultimately shed light on the physiological functions that this receptor plays in the kidney and liver. NIH/NCATS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Uncovering the role of GPR39 in the kidney

G protein-coupled receptors (GPCRs) constitute the largest class of proteins in the mammalian genome. Upon activation by a specific ligand, these receptors initiate downstream cellular responses that regulate various physiological processes. One such receptor under investigation in our lab is the ghrelin family receptor GPR39. Unlike its relatives, GPR39 does not respond to peptide hormones or neuropeptides. Initial studies suggested zinc as the endogenous ligand, but recent research indicates that zinc functions as an allosteric potentiator for another unidentified endogenous ligand. To date, synthetic agonists for GPR39 have revealed functions for GPR39 in the heart, bone, skin, pancreas, and gastrointestinal tract. However, GPR39’s role in renal physiology is currently unknown, despite relatively high kidney expression. To address this gap in knowledge, we first worked to localize GPR39 within the kidney. As a reliable antibody for GPR39 is not available, we utilized a combination of RNAScope (Gpr39) and immunofluorescence (AQP2). We find that GPR39 is expressed in the renal collecting duct, with the highest expression in AQP2-positive cells (principal cells) in the inner medullary collecting duct, and lesser expression in the cortical collecting duct. To determine if GPR39 is expressed apically or basolaterally, we then cloned GPR39 with a C-terminal EGFP tag and observed basolateral targeting of GPR39 in polarized MDCK cells grown on filters. In order to query the function of GPR39 in principal cells, we used murine principal kidney cortical collecting duct cells (mpkCCD). As others have shown, we find that mpkCCD express and traffc AQP2 to the apical plasma membrane only in the presence of the vasopressin analog dDAVP. Furthermore, we observe that treatment of mpkCCD with the GPR39-specific agonist cpd1324 induces internalization of AQP2 into cytoplasmic vesicles, even in the continued presence of dDAVP. Under vehicle conditions (dDAVP + vehicle), 80.9±7.7% (mean±SD) of AQP2 stain colocalized with an apical membrane marker (WGA staining). In contrast, when mpkCCD cells were co-treated ddAVP+cpd1324, only 58.3±6.02% of AQP2 stain colocalized with the apical membrane (p < 0.001, t-test). These results indicate that GPR39 activation may act to antagonize vasopressin-induced AQP2 traffcking in mpkCCD cells. NHLBIT32HL007534 (MK) and American Heart Association Established Investigator Award (JLP). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.