Development and tissue specific expression of RAPGEF1 (C3G) transcripts having exons encoding disordered segments with predicted regulatory function.

BACKGROUND: The ubiquitously expressed Guanine nucleotide exchange factor, RAPGEF1 (C3G), is essential for early development of mouse embryos. It functions to regulate gene expression and cytoskeletal reorganization, thereby controlling cell proliferation and differentiation. While multiple transcripts have been predicted, their expression in mouse tissues has not been investigated in detail. METHODS & RESULTS: Full length RAPGEF1 isoforms primarily arise due to splicing at two hotspots, one involving exon-3, and the other involving exons 12-14 incorporating amino acids immediately following the Crk binding region of the protein. These isoforms vary in expression across embryonic and adult organs. We detected the presence of unannotated, and unpredicted transcripts with incorporation of cassette exons in various combinations, specifically in the heart, brain, testis and skeletal muscle. Isoform switching was detected as myocytes in culture and mouse embryonic stem cells were differentiated to form myotubes, and embryoid bodies respectively. The cassette exons encode a serine-rich polypeptide chain, which is intrinsically disordered, and undergoes phosphorylation. In silico structural analysis using AlphaFold indicated that the presence of cassette exons alters intra-molecular interactions, important for regulating catalytic activity. LZerD based docking studies predicted that the isoforms with one or more cassette exons differ in interaction with their target GTPase, RAP1A. CONCLUSIONS: Our results demonstrate the expression of novel RAPGEF1 isoforms, and predict cassette exon inclusion as an additional means of regulating RAPGEF1 activity in various tissues and during differentiation.

Generation of B7-H3 isoform regulated by ANXA2/NSUN2/YBX1 axis in human glioma.

In recent years, in the development of emerging immunotherapy, B7-H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)-T target against glioma and other tumours, and aroused extensive attention. However, B7-H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7-H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7-H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5-methylcytosine reader Y-box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7-H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy.

Latrophilins as Downstream Effectors of Androgen Receptors including a Splice Variant, AR-V7, Induce Prostate Cancer Progression.

Latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin (LTX) is known to bind, remain largely uncharacterized in neoplastic diseases. In the present study, we aimed to determine the role of LPHNs in the progression of prostate cancer. We assessed the actions of LPHNs, including LPHN1, LPHN2, and LPHN3, in human prostate cancer lines via their ligand (e.g., α-LTX, FLRT3) treatment or shRNA infection, as well as in surgical specimens. In androgen receptor (AR)-positive LNCaP/C4-2/22Rv1 cells, dihydrotestosterone considerably increased the expression levels of LPHNs, while chromatin immunoprecipitation assay revealed the binding of endogenous ARs, including AR-V7, to the promoter region of each LPHN. Treatment with α-LTX or FLRT3 resulted in induction in the cell viability and migration of both AR-positive and AR-negative lines. α-LTX and FLRT3 also enhanced the expression of Bcl-2 and phosphorylated forms of JAK2 and STAT3. Meanwhile, the knockdown of each LPHN showed opposite effects on all of those mediated by ligand treatment. Immunohistochemistry in radical prostatectomy specimens further showed the significantly elevated expression of each LPHN in prostate cancer, compared with adjacent normal-appearing prostate, which was associated with a significantly higher risk of postoperative biochemical recurrence in both univariate and multivariable settings. These findings indicate that LPHNs function as downstream effectors of ARs and promote the growth of androgen-sensitive, castration-resistant, or even AR-negative prostate cancer.

AI-Assisted Processing Pipeline to Boost Protein Isoform Detection.

Proteomics, the study of proteins within biological systems, has seen remarkable advancements in recent years, with protein isoform detection emerging as one of the next major frontiers. One of the primary challenges is achieving the necessary peptide and protein coverage to confidently differentiate isoforms as a result of the protein inference problem and protein false discovery rate estimation challenge in large data. In this chapter, we describe the application of artificial intelligence-assisted peptide property prediction for database search engine rescoring by Oktoberfest, an approach that has proven effective, particularly for complex samples and extensive search spaces, which can greatly increase peptide coverage. Further, it illustrates a method for increasing isoform coverage by the PickedGroupFDR approach that is designed to excel when applied on large data. Real-world examples are provided to illustrate the utility of the tools in the context of rescoring, protein grouping, and false discovery rate estimation. By implementing these cutting-edge techniques, researchers can achieve a substantial increase in both peptide and isoform coverage, thus unlocking the potential of protein isoform detection in their studies and shedding light on their roles and functions in biological processes.

Insights into Actin Isoform-Specific Interactions with Myosin via Computational Analysis.

Actin, which plays a crucial role in cellular structure and function, interacts with various binding proteins, notably myosin. In mammals, actin is composed of six isoforms that exhibit high levels of sequence conservation and structural similarity overall. As a result, the selection of actin isoforms was considered unimportant in structural studies of their binding with myosin. However, recent high-resolution structural research discovered subtle structural differences in the N-terminus of actin isoforms, suggesting the possibility that each actin isoform may engage in specific interactions with myosin isoforms. In this study, we aimed to explore this possibility, particularly by understanding the influence of different actin isoforms on the interaction with myosin 7A. First, we compared the reported actomyosin structures utilizing the same type of actin isoforms as the high-resolution filamentous skeletal α-actin (3.5 Å) structure elucidated using cryo-EM. Through this comparison, we confirmed that the diversity of myosin isoforms leads to differences in interaction with the actin N-terminus, and that loop 2 of the myosin actin-binding sites directly interacts with the actin N-terminus. Subsequently, with the aid of multiple sequence alignment, we observed significant variations in the length of loop 2 across different myosin isoforms. We predicted that these length differences in loop 2 would likely result in structural variations that would affect the interaction with the actin N-terminus. For myosin 7A, loop 2 was found to be very short, and protein complex predictions using skeletal α-actin confirmed an interaction between loop 2 and the actin N-terminus. The prediction indicated that the positively charged residues present in loop 2 electrostatically interact with the acidic patch residues D24 and D25 of actin subdomain 1, whereas interaction with the actin N-terminus beyond this was not observed. Additionally, analyses of the actomyosin-7A prediction models generated using various actin isoforms consistently yielded the same results regardless of the type of actin isoform employed. The results of this study suggest that the subtle structural differences in the N-terminus of actin isoforms are unlikely to influence the binding structure with short loop 2 myosin 7A. Our findings are expected to provide a deeper understanding for future high-resolution structural binding studies of actin and myosin.

Splicing variants of versican in CD133+/CD44+ prostate cancer stem cells.

A cancer mass is composed of a heterogeneous group of cells, a small part of which constitutes the cancer stem cells since they are less differentiated and have a high capacity to develop cancer. Versican is an extracellular matrix protein located in many human tissues. The mRNA of versican has been shown to have "splicing patterns" as detected by RT-PCR, northern blot analysis, and cDNA sequencing. Based on this knowledge this study aims to reveal the splice variants of versican molecules, which are thought to be involved in the pathogenesis of the DU-145 human prostatic carcinoma cell line and prostatic cancer stem cells isolated from this cell line. In this study, RWPE-1 normal prostatic and DU-145 human prostate cancer cell lines have been used. Prostatic cancer stem cells and the remaining group of non-prostatic-cancer stem cells (bulk population) were isolated according to their CD133+/CD44+. RNA was isolated in all groups, and sequence analysis was accomplished for splicing variants by Illumina NextSeq 500 sequencing system. The results were analyzed by bioinformatic evaluation. As five isoforms of the versican gene in the differential transcript expression are analyzed, it was observed that a significant change was only found in the isoforms Versican 0 and Versican 1. In this study, we explored the function of this molecule which we think to be effective in cancer progression, and suggested that more valuable results can be obtained after the accomplishment of in vivo experiments.

Combining Native Mass Spectrometry and Proteomics to Differentiate and Map the Metalloform Landscape in Metallothioneins.

Within the intricate landscape of the proteome, approximately 30% of all proteins bind metal ions. This repertoire is even larger when considering all the different forms of a protein, known as proteoforms. Here, we propose the term "metalloforms" to refer to different structural or functional variations of a protein resulting from the binding of various hetero- or homogeneous metal ions. Using human Cu(I)/Zn(II)-metallothionein-3 as a representative model, we developed a chemical proteomics strategy to simultaneously differentiate and map Zn(II) and Cu(I) metal binding sites. In the first labeling step, N-ethylmaleimide reacts with Cysteine (Cys), resulting in the dissociation of all Zn(II) ions while Cu(I) remains bound to the protein. In the second labeling step, iodoacetamide is utilized to label Cu(I)-bound Cys residues. Native mass spectrometry (MS) was used to determine the metal/labeling protein stoichiometries, while bottom-up/top-down MS was used to map the Cys-labeled residues. Next, we used a developed methodology to interrogate an isolated rabbit liver metallothionein fraction containing three metallothionein-2 isoforms and multiple Cd(II)/Zn(II) metalloforms. The approach detailed in this study thus holds the potential to decode the metalloproteoform diversity within other proteins.

CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes.

The inhibitors, CK-666 and CK-869, are widely used to probe the function of Arp2/3 complex mediated actin nucleation in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 also only inhibits Arp3- but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.

A method for rapid and reliable quantification of VEGF-cell binding activity.

Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.

Functional Characterization of the Human BRCA1 ∆11 Splicing Isoforms in Yeast.

BRCA1, a crucial tumor suppressor gene, has several splicing isoforms, including Δ9-11, Δ11, and Δ11q, which lack exon 11, coding for significant portions of the protein. These isoforms are naturally present in both normal and cancerous cells, exhibiting altered activity compared to the full-length BRCA1. Despite this, the impact on cancer risk of the germline intronic variants promoting the exclusive expression of these Δ11 isoforms remains uncertain. Consequently, they are classified as variants of uncertain significance (VUS), posing challenges for traditional genetic classification methods due to their rarity and complexity. Our research utilizes a yeast-based functional assay, previously validated for assessing missense BRCA1 variants, to compare the activity of the Δ11 splicing isoforms with known pathogenic missense variants. This approach allows us to elucidate the functional implications of these isoforms and determine whether their exclusive expression could contribute to increased cancer risk. By doing so, we aim to provide insights into the pathogenic potential of intronic VUS-generating BRCA1 splicing isoforms and improve the classification of BRCA1 variants.

The CD44s Isoform is a Potential Biomarker for Predicting Craniopharyngioma Recurrence in Children.

Adamantinomatous craniopharyngioma (ACP) is an intracranial tumor considered partly malignant due to its ability to infiltrate surrounding structures and tendency to relapse despite radical resection. CD44 is a known stem cell marker in ACP and is upregulated in cell clusters of invasive ACP protrusions; however, the functions of its alternative splicing isoform variants, CD44s and CD44v1-10, have not yet been studied in terms of ACP recurrence, despite their confirmed roles in cancer development and progression. In this study, we first confirmed the difference in total CD44 expression between samples from patients who experienced relapse and those from patients who did not. Moreover, our findings showed that, in recurrent samples, the predominant isoform expressed was CD44s, which might indicate its significance in predicting ACP recurrence. The association between increased CD44 expression and recurrence may lead to the development of prognostic markers of ACP aggressiveness and relapse potential; however, further studies are needed to clarify the exact mechanism of CD44 expression.

Evaluation of androgen receptor and androgen receptor splice-variant 7 in bladder cancer; a novel approach into an ancient topic.

PURPOSE: The contribution of androgen receptors (AR) on bladder cancer has been demonstrated in pre-clinical studies, however in clinical studies, only the canonical AR (AR-FL) protein was measured by immunohistochemistry and conflicting results were obtained. To get better insight into the alterations of AR signalling, we used western blotting (WB) method and simultaneously measured both mRNA and protein levels of AR-FL and AR-V7. METHODS: 23 naive non-muscle invasive bladder cancer patients and 12 healthy individuals were included. AR-FL protein, AR-FL mRNA, AR-V7 protein and AR-V7 mRNA levels were quantitatively measured by WB and qRT-PCR. RESULTS: While AR-FL protein and AR-V7 mRNA were significantly higher in bladder cancer, AR-FL mRNA and AR-V7 protein were lower. AR-V7 mRNA level was higher in patients with tumour size over 3 cm and AR-FL protein was higher in single tumours (p < 0,005). The small sampling size and the inclusion of only male participants were the main limitations. CONCLUSIONS: The increase of AR-FL protein in bladder cancer supports the contribution of the AR pathway in bladder cancer. The presence of high AR-FL protein despite low mRNA levels may be due to a disruption in post-transcriptional regulatory mechanisms. AR-V7 was demonstrated for the first time in bladder tissue and found significantly different in bladder cancer tissues. Our study reached new and valuable findings and will shed light on the studies that aim to clarify the role of the AR pathway in bladder cancer.

Isoform-specific sequestration of protein kinase A fine-tunes intracellular signaling during heat stress.

Protein kinase A (PKA) is a conserved kinase crucial for fundamental biological processes linked to growth, development, and metabolism. The PKA catalytic subunit is expressed as multiple isoforms in diverse eukaryotes; however, their contribution to ensuring signaling specificity in response to environmental cues remains poorly defined. Catalytic subunit activity is classically moderated via interaction with an inhibitory regulatory subunit. Here, a quantitative mass spectrometry approach is used to examine heat-stress-induced changes in the binding of yeast Tpk1-3 catalytic subunits to the Bcy1 regulatory subunit. We show that Tpk3 is not regulated by Bcy1 binding but, instead, is deactivated upon heat stress via reversible sequestration into cytoplasmic granules. These "Tpk3 granules" are enriched for multiple PKA substrates involved in various metabolic processes, with the Hsp42 sequestrase required for their formation. Hence, regulated sequestration of Tpk3 provides a mechanism to control isoform-specific kinase signaling activity during stress conditions.

Unraveling the non-fitness status of NK cells: Examining the NKp30 receptor and its isoforms distribution in HIV/HCV coinfected patients.

BACKGROUND: HIV/HCV coinfection is associated with a rapid progression to liver damage. Specifically, NK cell population dysregulation is of particular interest, as these cells have been shown to block HCV replication effectively and have an anti-fibrogenic activity. The NKp30 receptor is linked to tumor cell lysis and has a crucial role during viral infections. In the present study, we determined the subpopulations of NK cells based on CD56 and CD16 expression, NKp30 receptor expression, its isoforms A, B, and C, along with the cytotoxicity molecules in patients with HIV/HCV. RESULTS: evidenced by the APRI and FIB-4 indices, the HCV-infected patients presented greater liver damage than the HIV and HIV/HCV groups. The HCV group presented a decreased expression of NKp30 isoform A, and NK cell frequency was not different between groups; however, CD56brigth subpopulation, NKp30 receptor, and CD247 adaptor chain were decreased in HIV/HCV patients; further, we described increased levels of soluble IL-8, IL-10, IL-12, and IL-23 in the serum of HIV/HCV patients. CONCLUSIONS: HCV and HIV/HCV patients have multiple parameters of non-fitness status in NK cells; awareness of these dysfunctional immunological parameters in HIV/HCV and HCV patients can elucidate possible novel therapeutics directed towards the improvement of NK cell fitness status, in order to improve their function against liver damage.

The structure and function of multifunctional protein ErbB3 binding protein 1 (Ebp1) and its role in diseases.

ErbB3-binding protein 1(Ebp1) has two isoforms, p42 Ebp1 and p48 Ebp1, both of which can regulate cell growth and differentiation. But these isoforms often have opposite effects, including contradictory roles in regulation of cell growth in different tissues and cells. P48 Ebp1 belongs to the full-length sequence, while conformational changes in the crystal structure of p42 Ebp1 reveals a lack of an α helix at the amino terminus. Due to the differences in the structures of these two isoforms, they have different binding partners and protein modifications. Ebp1 can function as both an oncogene and a tumor suppressor factor. However, the underlying mechanisms by which these two isoforms exert opposite functions are still not fully understood. In this review, we summarize the genes and the structures of protein of these two isoforms, protein modifications, binding partners and the association of different isoforms with diseases.

Assessment of EN-RAGE, sRAGE, and its isoforms: cRAGE, esRAGE in obese patients treated by moderate caloric restriction combined with physical activity conducted in hospital condition.

BACKGROUND: AGEs, their receptor (RAGE), and the extracellular newly identified receptor for AGEs product-binding protein (EN-RAGE) are implicated in the pathogenesis of inflammation. AIM: We analyzed serum EN-RAGE, soluble RAGE (sRAGE), and their isoforms: endogenous secretory - esRAGE and cleaved - cRAGE concentrations in lean controls (n = 74) and in patients with obesity (n = 71) treated for three weeks with moderate calorie restriction (CR) combined with physical activity in a hospital condition. METHODS: Using the ELISA method, serum sRAGE, esRAGE, and EN-RAGE were measured before and after CR. RESULTS: The serum level of sRAGE and esRAGE in patients with obesity was lower than that in non-obese individuals, contrary to cRAGE. EN-RAGE concentration was about three times higher in obese patients. Gradually, a rise in BMI resulted in sRAGE, esRAGE reduction, and EN-RAGE increase. The sRAGE concentration was sex-dependent, indicating a higher value in lean men. A moderate negative correlation was observed between BMI and all RAGE isoforms, whereas EN-RAGE displays a positive correlation. CR resulted in an expected decrease in anthropometric, metabolic, and proinflammatory parameters and EN-RAGE, but no RAGE isoforms. The ratio EN-RAGE/sRAGE was higher in obese humans than in control and was not modified by CR. CONCLUSION: Obesity decreases sRAGE and esRAGE and increases EN-RAGE concentration. Moderate CR and physical activity by decreasing inflammation reduces EN-RAGE but is insufficient to increase sRAGE and esRAGE to the extent observed in lean patients. EN-RAGE instead of sRAGE could be helpful to indicate a better outcome of moderate dietary intervention in obese subjects.

Androgen Receptor Splice Variant 7 in Asian Patients With Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Androgen receptor splice variant 7 (ARV-7) is a resistance mechanism to hormonal therapy in metastatic castrate-resistant prostate cancer (mCRPC). It has been associated with poor outcomes. On progression to castrate resistance, ARV-7 positivity has been identified in global populations at an incidence of 17.8%-28.8%. Here, we characterize the incidence of ARV-7 positivity in Asian patients with mCRPC in a prospective fashion and evaluate its implications on treatment outcomes. METHODS: Patients with mCRPC from multiple centers in Southeast and East Asia were enrolled in a prospective manner before initiation of androgen receptor signaling inhibitors or docetaxel. ARV-7 status was evaluated at baseline with three commercially available assays: AdnaTest Prostate Cancer platform, Clearbridge method, and IBN method. Clinical outcomes at progression were assessed. The primary end point of this study was prevalence of ARV-7 positivity; secondary end points were incidence of ARV-7 positivity, prostate specific antigen (PSA) response rate, PSA progression-free survival (PFS), and overall survival (OS). RESULTS: A total of 102 patients with a median age of 72 years at enrollment participated. Overall, an incidence of ARV-7 positivity of between 14.3% and 33.7% in Asian patients with mCRPC was demonstrated depending on the assay used. Patients found to have ARV-7 positivity at enrollment had a numerically worse PSA PFS compared with ARV-7 negative patients. CONCLUSION: In this study, the incidence of ARV-7 positivity in Asian patients with mCRPC was shown to be similar to the global population. Patients with ARV-7 positivity appear to have more aggressive disease with numerically worse PSA PFS and OS. Further prospective studies are needed to fully characterize the relationship that ARV-7 positivity has on prognosis of Asian patients with mCRPC.

Canonical and non-canonical functions of p53 isoforms: potentiating the complexity of tumor development and therapy resistance.

Full-length p53 (p53α) plays a pivotal role in maintaining genomic integrity and preventing tumor development. Over the years, p53 was found to exist in various isoforms, which are generated through alternative splicing, alternative initiation of translation, and internal ribosome entry site. p53 isoforms, either C-terminally altered or N-terminally truncated, exhibit distinct biological roles compared to p53α, and have significant implications for tumor development and therapy resistance. Due to a lack of part and/or complete C- or N-terminal domains, ectopic expression of some p53 isoforms failed to induce expression of canonical transcriptional targets of p53α like CDKN1A or MDM2, even though they may bind their promoters. Yet, p53 isoforms like Δ40p53α still activate subsets of targets including MDM2 and BAX. Furthermore, certain p53 isoforms transactivate even novel targets compared to p53α. More recently, non-canonical functions of p53α in DNA repair and of different isoforms in DNA replication unrelated to transcriptional activities were discovered, amplifying the potential of p53 as a master regulator of physiological and tumor suppressor functions in human cells. Both regarding canonical and non-canonical functions, alternative p53 isoforms frequently exert dominant negative effects on p53α and its partners, which is modified by the relative isoform levels. Underlying mechanisms include hetero-oligomerization, changes in subcellular localization, and aggregation. These processes ultimately influence the net activities of p53α and give rise to diverse cellular outcomes. Biological roles of p53 isoforms have implications for tumor development and cancer therapy resistance. Dysregulated expression of isoforms has been observed in various cancer types and is associated with different clinical outcomes. In conclusion, p53 isoforms have expanded our understanding of the complex regulatory network involving p53 in tumors. Unraveling the mechanisms underlying the biological roles of p53 isoforms provides new avenues for studies aiming at a better understanding of tumor development and developing therapeutic interventions to overcome resistance.

P2X7 Variants in Pathophysiology.

P2X7 receptor activation by extracellular adenosine triphosphate (eATP) modulates different intracellular pathways, including pro-inflammatory and tumor-promoting cascades. ATP is released by cells and necrotic tissues during stressful conditions and accumulates mainly in the inflammatory and tumoral microenvironments. As a consequence, both the P2X7 blockade and agonism have been proposed as therapeutic strategies in phlogosis and cancer. Nevertheless, most studies have been carried out on the WT fully functional receptor variant. In recent years, the discovery of P2X7 variants derived by alternative splicing mechanisms or single-nucleotide substitutions gave rise to the investigation of these new P2X7 variants' roles in different processes and diseases. Here, we provide an overview of the literature covering the function of human P2X7 splice variants and polymorphisms in diverse pathophysiological contexts, paying particular attention to their role in oncological and neuroinflammatory conditions.

Analysis of the TID-I and TID-L Splice Variants' Expression Profile under In Vitro Differentiation of Human Mesenchymal Bone Marrow Cells into Osteoblasts.

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants (TID-L, TID-I) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1. Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1. However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.