Protein Cellular Localization Research Articles

Chitin Deacetylase Homologous Gene cda Contributes to Development and Aflatoxin Synthesis in Aspergillus flavus.

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.

SCFFBXW11 Complex Targets Interleukin-17 Receptor A for Ubiquitin-Proteasome-Mediated Degradation.

Interleukin-17 (IL-17) is a pro-inflammatory cytokine that participates in innate and adaptive immune responses and plays an important role in host defense, autoimmune diseases, tissue regeneration, metabolic regulation, and tumor progression. Post-translational modifications (PTMs) are crucial for protein function, stability, cellular localization, cellular transduction, and cell death. However, PTMs of IL-17 receptor A (IL-17RA) have not been investigated. Here, we show that human IL-17RA was targeted by F-box and WD repeat domain-containing 11 (FBXW11) for ubiquitination, followed by proteasome-mediated degradation. We used bioinformatics tools and biochemical techniques to determine that FBXW11 ubiquitinated IL-17RA through a lysine 27-linked polyubiquitin chain, targeting IL-17RA for proteasomal degradation. Domain 665-804 of IL-17RA was critical for interaction with FBXW11 and subsequent ubiquitination. Our study demonstrates that FBXW11 regulates IL-17 signaling pathways at the IL-17RA level.

Expression of HIF‑α and their association with clinicopathological parameters in clinical renal cell carcinoma.

This study aimed to assess the cellular localization and expression levels of hypoxia-inducible factor (HIF) -α proteins (specifically HIF-1α, HIF-2α, and HIF-3α) that play a role in the hypoxia pathway and to determine their correlation with clinicopathological parameters and patient survival in renal cell carcinoma (RCC). Tissue microarray (TMA) with cores from 150 clear cell RCCs and 31 non-ccRCC samples. HIF-1α, HIF-2α, and HIF-3α antibodies were used for immunohistochemistry (IHC) of TMA to evaluate the cellular localization and expression levels of HIF-α proteins, specifically in relation to the hypoxia pathway. The expression levels of the HIF-α proteins were higher in the nucleus than in the cytoplasm. Furthermore, the nuclear expression levels of all HIF-α proteins were significantly higher in clear cell RCC (ccRCC) than in non-ccRCC. Cytoplasmic HIF-3α expression was also higher in ccRCC than in non-ccRCC, whereas cytoplasmic HIF-1α and HIF-2α expression levels were similar between the different RCC types. In ccRCC, nuclear HIF-1α expression levels correlated with both nuclear HIF-2α and HIF-3α levels, whereas cytoplasmic HIF-3α expression levels were associated with HIF-1α only.In non-ccRCC, there was a positive correlation observed between nuclear HIF-1α and HIF-3α expression, but no correlation was found with HIF-2α. In patients with ccRCC, the nuclear expressions of HIF-1α and HIF-3α was significantly associated with cancer-specific survival (CSS) in univariate analysis. This association was no longer evident in multivariate analysis. Notably, there was no correlation observed between nuclear HIF-2α expression and CSS in these patients. In contrast, cytoplasmic expression levels showed no association with CSS. The expression levels of the three primary HIF-α proteins were found to be higher in the nucleus than in the cytoplasm. Furthermore, the results indicated that HIF-3α and HIF-1α expression levels were significant univariate factors associated with CSS in patients with clear cell RCC. These results highlight the critical role that HIF-3α and HIF-1α play in the hypoxia pathway.

Phloretin Inhibits the Proliferation of Breast Cancer Cells Through the Down-regulation of Estrogen Receptor α.

Phloretin is a natural flavonoid compound found in some plants, such as apples and pears, as well as in the bark of apple trees. Phloretin has been shown to have inhibitory effects on glucose transporters in cells and can potentially inhibit the growth of cancer cells. However, the mechanism by which phloretin regulates the expression of estrogen receptor alpha (ERα), a key transcription factor in breast cancer, is still unclear. This study investigated how phloretin affects the growth of ERα positive human breast cancer cells. The growth of breast cancer cell lines, including MCF7 and T47D, was examined using cell proliferation and colony formation assays. Western blotting and semi-quantitative RT-PCR were used to examine protein and mRNA levels, respectively. Localization of cellular proteins was analyzed using subcellular fractionation. Transient transfection and reported gene assays were used to elucidate the impact of phloretin on cell proliferation and ERα transactivation. Phloretin decreased ERα expression at the mRNA and protein levels in MCF7 and T47D cells. It also inhibited the binding of ERα to the estrogen response element present in the promoter of target genes. Moreover, treatment with phloretin inhibited the expression of cyclin D1 and breast cancer marker gene pS2, which are known ERα target genes. Consequently, it inhibited the growth of ERα-positive human breast cancer cells. Furthermore, inhibition of breast cancer growth by phloretin was found to be mediated through both the ERα and ERK1/ERK2 pathways. Phloretin, a dihydrochalcone extracted from natural sources, exhibits the ability to regulate ERα function and suppress breast cancer cell proliferation.

Deep-Learning Uncovers certain CCM Isoforms as Transcription Factors.

Cerebral Cavernous Malformations (CCMs) are brain vascular abnormalities associated with an increased risk of hemorrhagic strokes. Familial CCMs result from autosomal dominant inheritance involving three genes: KRIT1 (CCM1), MGC4607 (CCM2), and PDCD10 (CCM3). CCM1 and CCM3 form the CCM Signal Complex (CSC) by binding to CCM2. Both CCM1 and CCM2 exhibit cellular heterogeneity through multiple alternative spliced isoforms, where exons from the same gene combine in diverse ways, leading to varied mRNA transcripts. Additionally, both demonstrate nucleocytoplasmic shuttling between the nucleus and cytoplasm, suggesting their potential role in gene expression regulation as transcription factors (TFs). Due to the accumulated data indicating the cellular localization of CSC proteins in the nucleus and their interaction with progesterone receptors, which serve dual roles as both cellular signaling components and TFs, a question has arisen regarding whether CCMs could also function in both capacities like progesterone receptors. To investigate this potential, we employed our proprietary deep-learning (DL)-based algorithm, specifically utilizing a biased-Support Vector Machine (SVM) model, to explore the plausible cellular function of any of the CSC proteins, particularly focusing on CCM gene isoforms with nucleocytoplasmic shuttling, acting as TFs in gene expression regulation. Through a comparative DL-based predictive analysis, we have effectively discerned a collective of 11 isoforms across all CCM proteins (CCM1-3). Additionally, we have substantiated the TF functionality of 8 isoforms derived from CCM1 and CCM2 proteins, marking the inaugural identification of CCM isoforms in the role of TFs. This groundbreaking discovery directly challenges the prevailing paradigm, which predominantly emphasizes the involvement of CSC solely in endothelial cellular functions amid various potential cellular signal cascades during angiogenesis.

BRCC36 associates with FLT3-ITD to regulate its protein stability and intracellular signaling in acute myeloid leukemia.

Fms-like tyrosine kinase-3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal-tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3-ITD and FLT3-TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3-ITD or -TKD differently. We found that BRCA1/BRCA2-containing complex subunit 36 (BRCC36), a specific K63-linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3-ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3-ITD, which may shed light on developing a novel therapeutic approach for AML.

PPSNO: A Feature-Rich SNO Sites Predictor by Stacking Ensemble Strategy from Protein Sequence-Derived Information.

The protein S-nitrosylation (SNO) is a significant post-translational modification that affects the stability, activity, cellular localization, and function of proteins. Therefore, highly accurate prediction of SNO sites aids in grasping biological function mechanisms. In this document, we have constructed a predictor, named PPSNO, forecasting protein SNO sites using stacked integrated learning. PPSNO integrates multiple machine learning techniques into an ensemble model, enhancing its predictive accuracy. First, we established benchmark datasets by collecting SNO sites from various sources, including literature, databases, and other predictors. Second, various techniques for feature extraction are applied to derive characteristics from protein sequences, which are subsequently amalgamated into the PPSNO predictor for training. Five-fold cross-validation experiments show that PPSNO outperformed existing predictors, such as PSNO, PreSNO, pCysMod, DeepNitro, RecSNO, and Mul-SNO. The PPSNO predictor achieved an impressive accuracy of 92.8%, an area under the curve (AUC) of 96.1%, a Matthews correlation coefficient (MCC) of 81.3%, an F1-score of 85.6%, an SN of 79.3%, an SP of 97.7%, and an average precision (AP) of 92.2%. We also employed ROC curves, PR curves, and radar plots to show the superior performance of PPSNO. Our study shows that fused protein sequence features and two-layer stacked ensemble models can improve the accuracy of predicting SNO sites, which can aid in comprehending cellular processes and disease mechanisms. The codes and data are available at https://github.com/serendipity-wly/PPSNO .

SUMO Sites Prediction in Human Transcription Factors Involved in Hypoxia induced Cardiac Illnesses

Protein SUMOylation is a reversible and well knownpost-translational modificationprocess of the cells. It may change a protein's cellular location, interactions, and possible structural shape before it develops to carry out its basic functions.Also, it decides the binding of transcription factors and DNA binding proteins tochromatin in addition to various cis and trans regulatory factors. Alterations in protein SUMOylation have been linked with a variety of disorders and developmental anomalies.Tentative approaches to identify SUMO binding sites are challenging due todynamic nature of the SUMOylation processand various critical lab experimentswhich are involved very high cost.Therefore, the computational methodologies may guide the experimental identification of SUMOylation sites and provide insights for improving comprehensionofSUMOylation mechanism in the cells.In this study, we identify the SUMO binding sites in transcription factors that are actively involved and have crucial roles in cardiac development andpathophysiology of the heart.A list of important transcription factors was preparedfrom thehuman transcription factor database.The GPS-SUMO, SUMO plot, and JASSA web serverswere used for the prediction of SUMO binding sites in cardiac transcription factors.We identified the SUMOylation of several novel, previously uncharacterized SUMO targetsthat are actively involved in thecardiovascular system.Thus, the present study may help to uncoverthe significance ofSUMO modificationin cardiac development and illnesses which creates a fresh avenue for future studies ontarget-specific SUMOylation for identification of novel therapeutic targets andmanagement strategies forhypoxia-induced cardiovascular disorders.

Automated identification of protein expression intensity and classification of protein cellular locations in mouse brain regions from immunofluorescence images.

Knowledge of protein expression in mammalian brains at regional and cellular levels can facilitate understanding of protein functions and associated diseases. As the mouse brain is a typical mammalian brain considering cell type and structure, several studies have been conducted to analyze protein expression in mouse brains. However, labeling protein expression using biotechnology is costly and time-consuming. Therefore, automated models that can accurately recognize protein expression are needed. Here, we constructed machine learning models to automatically annotate the protein expression intensity and cellular location in different mouse brain regions from immunofluorescence images. The brain regions and sub-regions were segmented through learning image features using an autoencoder and then performing K-means clustering and registration to align with the anatomical references. The protein expression intensities for those segmented structures were computed on the basis of the statistics of the image pixels, and patch-based weakly supervised methods and multi-instance learning were used to classify the cellular locations. Results demonstrated that the models achieved high accuracy in the expression intensity estimation, and the F1 score of the cellular location prediction was 74.5%. This work established an automated pipeline for analyzing mouse brain images and provided a foundation for further study of protein expression and functions.

Examining Phosphatases Through Immunofluorescent Microscopy.

Immunofluorescent microscopy enables the examination of cellular expression and localization of proteins. Cellular localization can often impact protein function, as certain molecular interactions occur in specific cellular compartments. Here we describe in detail the processes necessary for identifying phosphatases in the cell through immunofluorescent microscopy. Identification of phosphatase expression and localization could lead to the discovery of protein function in disease states along with potential substrates and binding partners.

Insights into the Cellular Localization and Functional Properties of TSPYL5 Protein.

In recent years, the role of liquid-liquid phase separation (LLPS) and intrinsically disordered proteins (IDPs) in cellular molecular processes has received increasing attention from researchers. One such intrinsically disordered protein is TSPYL5, considered both as a marker and a potential therapeutic target for various oncological diseases. However, the role of TSPYL5 in intracellular processes remains unknown, and there is no clarity even in its intracellular localization. In this study, we characterized the intracellular localization and exchange dynamics with intracellular contents of TSPYL5 and its parts, utilizing TSPYL5 fusion proteins with EGFP. Our findings reveal that TSPYL5 can be localized in both the cytoplasm and nucleoplasm, including the nucleolus. The nuclear (nucleolar) localization of TSPYL5 is mediated by the nuclear/nucleolar localization sequences (NLS/NoLS) identified in the N-terminal intrinsically disordered region (4-27 aa), while its cytoplasmic localization is regulated by the ordered NAP-like domain (198-382 aa). Furthermore, our results underscore the significant role of the TSPYL5 N-terminal disordered region (1-198 aa) in the exchange dynamics with the nucleoplasm and its potential ability for phase separation. Bioinformatics analysis of the TSPYL5 interactome indicates its potential function as a histone and ribosomal protein chaperone. Taken together, these findings suggest a significant contribution of liquid-liquid phase separation to the processes involving TSPYL5, providing new insights into the role of this protein in the cell's molecular life.

Identification of differentially expressed genes, signaling pathways and immune infiltration in postmenopausal osteoporosis by integrated bioinformatics analysis

BackgroundPostmenopausal osteoporosis is a systemic metabolic disorder typified by an imbalance in bone turnover, where bone resorption supersedes bone formation. This imbalance primarily arises from a decline in bone mass induced by estrogen deficiency, and an elevated risk of fractures resulting from degradation of bone microstructure. Despite recognizing these changes, the precise causative factors and potential molecular pathways remain elusive. In this study, we aimed to identify differentially expressed genes (DEGs), associated pathways, and the role of immune infiltration in osteoporosis, leveraging an integrated bioinformatics approach to shed light on potential underlying molecular mechanisms. MethodsWe retrieved the expression profiles of GSE230665 from the Gene Expression Omnibus database, comprising 15 femur samples, including 12 postmenopausal osteoporosis samples and 3 normal controls. From the aggregated microarray datasets, we derived differentially expressed genes (DEGs) for further bioinformatics analysis. We used WGCNA, analyzed DEGs, PPI, and conducted GO analysis to identify pivotal genes. We then used the CIBERSORT method to explore the degree of immune cell infiltration within femur specimens affected by postmenopausal osteoporosis. To probe into the relationship between pivotal genes and infiltrating immune cells, we conducted correlation analysis. ResultsWe identified a total of 12,204 DEGs. Among these, 12,157 were up-regulated, and 47 were down-regulated. GO and KEGG pathway analyses indicated that these DEGs predominantly targeted cellular protein localization activity and associated signaling pathways. The protein-protein interaction network highlighted four central hub-genes: RPL31, RPL34, EEF1G, and BPTF. Principal component analysis indicated a positive correlation between the expression of these genes and resting NK cells (as per CIBERSORT). In contrast, the expression of RPL31, RPL34, and EEF1G showed a negative correlation with T cells (gamma delta per CIBERSORT). ConclusionsImmune infiltration plays a role in the development of osteoporosis.

Tyrosine hydroxylase variants influence protein expression, cellular localization, stability, enzymatic activity and the physical interaction between tyrosine hydroxylase and GTP cyclohydrolase 1.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis catalyzing the tetrahydrobiopterin (BH4)-dependent hydroxylation of tyrosine to L-DOPA. Here, we analyzed 25 TH variants associated with various degrees of dopa-responsive dystonia and evaluate the effect of each variant on protein stability, activity and cellular localization. Furthermore, we investigated the physical interaction between TH and human wildtype (wt) GTP cyclohydrolase 1 (GTPCH) and the effect of variants on this interaction. Our in vitro results classify variants according to their resistance to proteinase K digestion into three groups (stable, intermediate, unstable). Based on their cellular localization, two groups of variants can be identified, variant group one with cytoplasmic distribution and variant group two forming aggregates. These aggregates do not correlate with loss of enzymatic activity but nevertheless might be a good target for molecular chaperones. Unfortunately, no obvious correlation between the half-life of a variant and its enzymatic activity or between solubility, stability and enzymatic activity of a given variant could be found. Excitingly, some variants disrupt the physical interaction between TH and human wildtype GTPCH, thereby interfering with enzymatic activity and offering new druggable targets for therapy. Taken together, our results highlight the importance of an in-depth molecular analysis of each variant in order to be able to classify groups of disease variants and to find specific therapies for each subgroup. Stand-alone in silico analyses predict less precise the effect of specific variants and should be combined with other in vitro analyses in cellular model systems.

Trichinella spiralis cathepsin L damages the tight junctions of intestinal epithelial cells and mediates larval invasion.

Cathepsin L, a lysosomal enzyme, participates in diverse physiological processes. Recombinant Trichinella spiralis cathepsin L domains (rTsCatL2) exhibited natural cysteine protease activity and hydrolyzed host immunoglobulin and extracellular matrix proteins in vitro, but its functions in larval invasion are unknown. The aim of this study was to explore its functions in T. spiralis invasion of the host's intestinal epithelial cells. RNAi significantly suppressed the expression of TsCatL mRNA and protein with TsCatL specific siRNA-302. T. spiralis larval invasion of Caco-2 cells was reduced by 39.87% and 38.36%, respectively, when anti-TsCatL2 serum and siRNA-302 were used. Mice challenged with siRNA-302-treated muscle larvae (ML) exhibited a substantial reduction in intestinal infective larvae, adult worm, and ML burden compared to the PBS group, with reductions of 44.37%, 47.57%, and 57.06%, respectively. The development and fecundity of the females from the mice infected with siRNA-302-treated ML was significantly inhibited. After incubation of rTsCatL2 with Caco-2 cells, immunofluorescence test showed that the rTsCatL2 gradually entered into the cells, altered the localization of cellular tight junction proteins (claudin 1, occludin and zo-1), adhesion junction protein (e-cadherin) and extracellular matrix protein (laminin), and intercellular junctions were lost. Western blot showed a 58.65% reduction in claudin 1 expression in Caco-2 cells treated with rTsCatL2. Co-IP showed that rTsCatL2 interacted with laminin and collagen I but not with claudin 1, e-cadherin, occludin and fibronectin in Caco-2 cells. Moreover, rTsCatL2 disrupted the intestinal epithelial barrier by inducing cellular autophagy. rTsCatL2 disrupts the intestinal epithelial barrier and facilitates T. spiralis larval invasion.

Expression profiles of glucocorticoid-inducible proteins in human papilloma virus-related oropharyngeal squamous cell carcinoma.

Human papillomavirus virus-related oropharyngeal squamous cell carcinoma (HPV-OPSCC) comprises a significant portion of head and neck cancers. Several glucocorticoid-inducible proteins play important roles in pathogenesis of some cancers but their status and roles in HPV-OPSCC remain elusive; these include the glucocorticoid-induced leucine zipper (GILZ), Annexin-A1 and serum glucocorticoid-regulated kinase-1 (SGK-1). We determined expression profiles of these proteins, using immunohistochemistry, in archived biopsy samples of patients diagnosed with HPV-OPSCC; samples of non-cancer oral lesions (e.g., hyperkeratosis) were used as controls. GILZ staining was primarily confined to nuclei of all tissues but, in HPV-OPSCC specimens, neoplastic cells exhibiting mitosis displayed prominent cytoplasmic GILZ expression. On the other hand, nuclear, cytoplasmic and membranous Annexin-A1 staining was observed in suprabasal cell layers of control specimens. A noted feature of the HPV-OPSCC specimens was few clusters of matured and differentiated nonbasaloid cells that showed prominent nuclear and cytoplasmic Annexin-A1 staining while the remainder of the tumor mass was devoid of staining. Cytoplasmic and nuclear staining for SGK-1 was prominent for control than PV-OPSCC specimens while staining for phosphorylated SGK-1 (pSGK-1; active) was prominent for cell membrane and cytoplasm of control specimens but HPV-OPSCC specimens showed mild and patchy nuclear and cytoplasmic staining. Semi-quantitative analysis of GILZ immunostaining indicated increased staining area but similar normalized staining for HPV-OPSCC compared to control specimens. By contrast, staining area and normalized staining were reduced for other proteins in HPV-OPSCC than control specimens. Our collective observations suggest differential cellular localization and expression of glucocorticoid-inducible proteins in HPV-OPSCC suggestive of different functional roles in pathogenesis of this condition.

Protein expression and localization of ABC transporters in pancreatic adenocarcinoma: Prognostic role of ABCC8

BackgroundATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients’ prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers – ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). MethodsProtein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. ResultsNo CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). ConclusionsThe study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.

3D Modelling and Bioinformatics Analysis of <i>Labeo catla</i> <i>Kisspeptin1</i> and <i>Kisspeptin2</i> Proteins

Here we characterized the Kisspeptin1 and Kisspeptin2 proteins of catla, Labeo catla ( Catla catla) - the second most important Indian major carp. The catla full-length Kisspeptin1 gene was 753 bp long which encoded a peptide of 116 AA (Amino Acids). The MW (Molecular Weight) and IP [Point Isoelectric (pl)] of catla Kisspeptin1 protein were observed to be 13371.28 kilodaltons (kDa) and 9.69, respectively. The predicted 3D structure of Kisspeptin1 protein showed a ROQF of 94.393%, and the RC Plot analysis showed that 90.35% residues were present in the most favourable region. There were 6 Serine, 2 Tyrosine and 6 Threonine P-sites, but no Ng-sites on the predicted protein were observed. Cellular localization of Kisspeptin1 protein was observed to be 31.6% in ECR, 30.8% in MR and 23 .1 % in NR. However, in the Kisspeptin2 gene, the full-length Kisspeptin2 gene was 583 bp which encoded a peptide of 125 AA. The catla Kisspeptin2 protein has an MW of 14504.60 kilodaltons (kDa) and an IP of 8.46. The predicted 3D structure of Kisspeptin2 protein showed a ROQF of 94.872% and the RC Plot analysis showed that 90.01 % residues are present in the most favoured region. There were 7 Threonine, 6 Serine, and 2 Tyrosine P-sites, but no Ng-sites were observed on the predicted protein. In contrast to Kisspeptin1 protein, 49.7% of Kisspeptin2 was observed to be present in the NR and 38.3% in the ER. The NJPT analysis showed that catla Kisspeptin1 and Kisspeptin2 amino acids showed a high similarity to Carassius auratus followed by Labeo rohita. The information generated from the present study will lay a solid infrastructure for grasp the role of Kisspeptin1 and Kisspeptin2 proteins in the NEC (Neuro-Endocrine Control) of the reproductive system in Labeo catla.

Impact of human ovarian tissue manipulation on follicles: evidence of a potential first wave of follicle activation during fertility preservation procedures.

The aim of this study was to investigate the impact of processing human ovarian tissue on follicle activation dynamics. Fresh ovarian tissue was retrieved from 9 women undergoing laparoscopic surgery for benign conditions. Biopsies from each patient were divided into 3 fragments, the first of which was immediately fixed in the operating room (T0) and the second and third just after processing at 25 (T25) and (T90) 90 min. To evaluate follicle activation, markers of the PI3K and Hippo signaling pathways were immunolabeled at each time point, targeting phospho-Akt (p-Akt) by immunohistochemistry and yes-associated protein (YAP) cellular localization in the granulosa cell layer by immunofluorescence. Four hundred forty primordial follicles were evaluated for p-Akt and 420 for YAP. Significantly stronger p-Akt expression was observed at T25 (23.01 ± 13.45%; p=0.04) and T90 (38.99 ± 25.21%; p<0.001) than at T0 (2.72 ± 3.35%). A significant nucleus-to-cytoplasm shift in YAP was detected at T25 (1.21 ± 0.25; p=0.015 compared to T0 (0.95 ± 0.09), while T90 (1.10 ± 0.16) values were similar to T25. Our data prove that ovarian tissue manipulation significantly impacts follicle dynamics by stimulating the PI3K and Hippo signaling pathways involved in primordial follicle activation. Further experimental evidence must nevertheless be gathered to understand and gain control of follicle activation mechanisms in non-physiological conditions (like ovarian tissue manipulation), in order to optimize fertility preservation and restoration strategies.

A+T rich interaction domain protein 3a (Arid3a) impairs Mertk-mediated efferocytosis in cholestasis

Macrophages are key elements in the pathogenesis of cholestatic liver diseases. Arid3a plays a prominent role in the biologic properties of hematopoietic stem cells, B lymphocytes and tumor cells, but its ability to modulate macrophage function during cholestasis remains unknown. Gene and protein expression and cellular localization were assessed by q-PCR, immunohistochemistry, immunofluorescence staining and flow cytometry. We generated myeloid-specific Arid3a knockout mice and established three cholestatic murine models. The transcriptome was analyzed by RNA-seq. A specific inhibitor of the Mertk receptor was used invitro and invivo. Promoter activity was determined by chromatin immunoprecipitation-seq against Arid3a and a luciferase reporter assay. In cholestatic murine models, myeloid-specific deletion of Arid3a alleviated cholestatic liver injury (accompanied by decreased accumulation of macrophages). Arid3a-deficient macrophages manifested a more reparative phenotype, which was eliminated by invitro treatment with UNC2025, a specific inhibitor of the efferocytosis receptor Mertk. Efferocytosis of apoptotic cholangiocytes was enhanced in Arid3a-deficient macrophages via upregulation of Mertk. Arid3a negatively regulated Mertk transcription by directly binding to its promoter. Targeting Mertk invivo effectively reversed the protective phenotype of Arid3a deficiency in macrophages. Arid3a was upregulated in hepatic macrophages and circulating monocytes in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Mertk was correspondingly upregulated and negatively correlated with Arid3a expression in PBC and PSC. Mertk+ cells were located in close proximity to cholangiocytes, while Arid3a+ cells were scattered among immune cells with greater spatial distances to hyperplastic cholangiocytes in PBC and PSC. Arid3a promotes cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes by macrophages during cholestasis. The Arid3a-Mertk axis is a promising novel therapeutic target for cholestatic liver diseases. Macrophages play an important role in the pathogenesis of cholestatic liver diseases. This study reveals that macrophages with Arid3a upregulation manifest a pro-inflammatory phenotype and promote cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes during cholestasis. Although we now offer a new paradigm to explain how efferocytosis is regulated in a myeloid cell autonomous manner, the regulatory effects of Arid3a on chronic liver diseases remain to be further elucidated.

Identification of EGF Receptor and Thrombospondin-1 as Endogenous Targets of ER-Associated Degradation Enhancer EDEM1 in HeLa Cells.

Secretory and membrane proteins are vital for cell activities, including intra- and intercellular communication. Therefore, protein quality control in the endoplasmic reticulum (ER) is an essential and crucial process for eukaryotic cells. Endoplasmic reticulum-associated degradation (ERAD) targets misfolded proteins during the protein maturation process in the ER and leads to their disposal. This process maintains the ER productive function and prevents misfolded protein stress (i.e., ER stress). The ERAD-stimulating factor ER degradation-enhancing α mannosidase-like 1 protein (EDEM1) acts on misfolded proteins to accelerate ERAD, thereby maintaining the productivity of the ER. However, the detail mechanism underlying the function of EDEM1 in ERAD is not completely understood due to a lack of established physiological substrate proteins. In this study, we attempted to identify substrate proteins for EDEM1 using siRNA. The matrix component thrombospondin-1 (TSP1) and epidermal growth factor receptor (EGFR) were identified as candidate targets of EDEM1. Their protein maturation status and cellular localization were markedly affected by knockdown of EDEM1. We also showed that EDEM1 physically associates with EGFR and enhances EGFR degradation via ERAD. Our data highlight the physiological role of EDEM1 in maintaining specific target proteins and provide a potential approach to the regulation of expression of clinically important proteins.