SH3 Domain Research Articles

Cellular spermine targets JAK signaling to restrain cytokine-mediated autoimmunity

Prolonged activation of the type I interferon (IFN-I) pathway leads to autoimmune diseases such as systemic lupus erythematosus (SLE). Metabolic regulation of cytokine signaling is critical for cellular homeostasis. Through metabolomics analyses of IFN-β-activated macrophages and an IFN-stimulated-response-element reporter screening, we identified spermine as a metabolite brake for Janus kinase (JAK) signaling. Spermine directly bound to the FERM and SH2 domains of JAK1 to impair JAK1-cytokine receptor interaction, thus broadly suppressing JAK1 phosphorylation triggered by cytokines IFN-I, IFN-II, interleukin (IL)-2, and IL-6. Peripheral blood mononuclear cells (PBMCs) from individuals with SLE showing decreased spermine concentrations exhibited enhanced IFN-I and lupus gene signatures. Spermine treatment attenuated autoimmune pathogenesis in SLE and psoriasis mice and reduced IFN-I signaling in monocytes from individuals with SLE. We synthesized a spermine derivative (spermine derivative 1 [SD1]) and showed that it had a potent immunosuppressive function. Our findings reveal spermine as a metabolic checkpoint for cellular homeostasis and a potential immunosuppressive molecule for controlling autoimmune disease.

Folding and Binding Kinetics of the Tandem of SH2 Domains from SHP2.

The SH2 domains of SHP2 play a crucial role in determining the function of the SHP2 protein. While the folding and binding properties of the isolated NSH2 and CSH2 domains have been extensively studied, there is limited information about the tandem SH2 domains. This study aims to elucidate the folding and binding kinetics of the NSH2-CSH2 tandem domains of SHP2 through rapid kinetic experiments, complementing existing data on the isolated domains. The results indicate that while the domains generally fold and unfold independently, acidic pH conditions induce complex scenarios involving the formation of a misfolded intermediate. Furthermore, a comparison of the binding kinetics of isolated NSH2 and CSH2 domains with the NSH2-CSH2 tandem domains, using peptides that mimic specific portions of Gab2, suggests a dynamic interplay between NSH2 and CSH2 in binding Gab2 that modulate the microscopic association rate constant of the binding reaction. These findings, discussed in the context of previous research on the NSH2 and CSH2 domains, enhance our understanding of the function of the SH2 domain tandem of SHP2.

Asciminib is a novel inhibitor of ABL1 and ABL2 gene fusions in ALL but requires the ABL SH3 domain for efficacy

High-risk Ph-like ALL includes genomic rearrangement of the ABL1 and ABL2 genes (collectively ABL-rearranged, ABLr), and novel treatments are required. For the first time, we demonstrate asciminib efficacy in ABLr ALL, but only when the ABL SH3 domain is present.

Structure and Dynamics of Drk-SH2 Domain and Its Site-Specific Interaction with Sev Receptor Tyrosine Kinase.

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.

Weighted single-step genome-wide association study for direct and maternal genetic effects associated with birth and weaning weights in sheep

Body weight is an important economic trait for sheep meat production, and its genetic improvement is considered one of the main goals in the sheep breeding program. Identifying genomic regions that are associated with growth-related traits accelerates the process of animal breeding through marker-assisted selection, which leads to increased response to selection. In this study, we conducted a weighted single-step genome-wide association study (WssGWAS) to identify potential candidate genes for direct and maternal genetic effects associated with birth weight (BW) and weaning weight (WW) in Baluchi sheep. The data used in this research included 13,408 birth and 13,170 weaning records collected at Abbas-Abad Baluchi Sheep Breeding Station, Mashhad-Iran. Genotypic data of 94 lambs genotyped by Illumina 50K SNP BeadChip for 54,241 markers were used. The proportion of variance explained by genomic windows was calculated by summing the variance of SNPs within 1 megabase (Mb). The top 10 window genomic regions explaining the highest percentages of additive and maternal genetic variances were selected as candidate window genomic regions associated with body weights. Our findings showed that for BW, the top-ranked genomic regions (1 Mb windows) explained 4.30 and 4.92% of the direct additive and maternal genetic variances, respectively. The direct additive genetic variance explained by the genomic window regions varied from 0.31 on chromosome 1 to 0.59 on chromosome 8. The highest (0.84%) and lowest (0.32%) maternal genetic variances were explained by genomic windows on chromosome 10 and 17, respectively. For WW, the top 10 genomic regions explained 6.38 and 5.76% of the direct additive and maternal genetic variances, respectively. The highest and lowest contribution of direct additive genetic variances were 1.37% and 0.42%, respectively, both explained by genomic regions on chromosome 2. For maternal effects on WW, the highest (1.38%) and lowest (0.41%) genetic variances were explained by genomic windows on chromosome 2. Further investigation of these regions identified several possible candidate genes associated with body weight. Gene ontology analysis using the DAVID database identified several functional terms, such as translation repressor activity, nucleic acid binding, dehydroascorbic acid transporter activity, growth factor activity and SH2 domain binding.

PTPN11variants in chronic myelomonocytic leukemia: Phenotypic and prognostic correlates.

6513 Background: PTPN11 (Ch12q24) is a proto-oncogene that codes for SHP2, a regulatory protein tyrosine phosphatase that impacts RAS/MAPK signaling. Germline variants have been described in juvenile myelomonocytic leukemia, while somatic variants have been associated with inferior survival in acute myeloid leukemia. PTPN11 variants are infrequent in chronic myelomonocytic leukemia (CMML), and their impact on disease phenotype and survival remains unclear. Methods: After IRB approval, 396 molecularly annotated CMML patients at Mayo Clinic were screened for somatic PTPN11 variants within 12 months of diagnosis while still in chronic phase disease. Overall (OS), acute myeloid leukemia (AML)-free survival (LFS), and predictors of survival were estimated using standard statistical measures. Results: 396 patients (median age 71 years; males 67%) with CMML were considered; pathogenic PTPN11 variants were seen in 12 (3%) with a median VAF of 20.5% (range: 4-50): A72T (n = 2), F285S (n = 2), and A72S (n = 2). Variants were most likely to be seen in the N-terminal SH2 domain (n = 7), followed by the PTP domain (n = 6), and the C-terminal SH2 domain (n = 1). PTPN11 variants clustered with CMML-2 (p = 0.02), higher absolute neutrophil count (p = 0.04), peripheral blood (p = 0.06) and bone marrow (p = 0.02) blast %, older age (p <0.01), and were less likely to have SRSF2 comutations (p = 0.02). After a median follow-up of 18 months (range 0-193), 253 (64%) deaths and 68 (17%) AML transformations were recorded. PTPN11 variants negatively and independently impacted both OS (median 13 vs. 31 months; p <0.01) and LFS (AML events: 42% (5/12) vs. 16% (63/384); p <0.01). In age-adjusted multivariate analysis restricted to genetic risk factors, survival was negatively affected by abnormal karyotype and mutations involving PTPN11, DNMT3A, and SETBP1, and positively by TET2 mutations. All but SETBP1 retained significance in the presence of additional independent risk factors, namely male sex (p <0.01) and leukocyte count (p <0.01); HR in the latter analysis was 4.3 for PTPN11, 2.8 for DNMT3A, and 1.6 for abnormal karyotype. A similar analysis for LFS identified, as risk factors, PTPN11 (p = 0.01), DNMT3A (p <0.01), and ASXL1 (p = 0.03) mutations, as well as bone marrow blast % (p <0.01), absolute monocyte count ≥10 (p = 0.02), and immature circulating myeloid cells (p <0.01). Conclusions: The current study identifies PTPN11 and DNMT3A mutations as independent risk factors for both OS and LFS in CMML. In regard to additional genetic risk factors, OS was positively affected by TET2 mutations and negatively by abnormal karyotype, while LFS was negatively affected by ASXL1 mutations.

Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ~5% of all human cancers worldwide, including most cervical cancer cases and a growing number of anogenital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited the oncogenic phenotype in vitro, whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we demonstrate that the LASP1 SH3 domain is essential for LASP1-mediated oncogenicity in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulates LASP1 mRNA levels by binding to its 3’UTR. Finally, we demonstrate that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival in cervical cancer, thus identifying a potential therapeutic target in these cancers.

Integrated Clinical Genotype-Phenotype Characteristics of STAT3-Mutated Myeloid Neoplasms.

STAT3 is a key transcription factor that mediates cancer progression through phosphorylation or gain-of-function mutations. STAT3 activation in myeloid neoplasms (MN) is primarily mediated through phosphorylation. STAT3 mutation has only rarely been reported in MNs. We assessed the clinicopathologic and molecular genetic features of 32 STAT3-mutated MNs. The frequency of STAT3 mutation in MNs was <0.5%. Twenty (62.5%) cases were classified as acute myeloid leukemia, 7 (21.9%) as myelodysplastic syndrome, and 5 (15.6%) as chronic myelomonocytic leukemia, but none as myeloproliferative neoplasms. STAT3 mutations occurred at initial diagnosis in 22 (88%) cases or at relapse or upon leukemic transformation. Clonal hierarchy analysis revealed that STAT3 mutations represented the dominant clone in 30% of acute myeloid leukemia cases but were subclonal in myelodysplastic syndrome and chronic myelomonocytic leukemia. Most were missense mutations located at the SH2 domain, Y640F being the most common. STAT3 mutation was accompanied by coexisting mutations in all cases, most frequently SRSF2, TET2, ASXL1, and SETBP1. STAT3 mutations were usually associated with morphologic dysplasia, increased blasts, and monosomy 7/del7q. With a median follow-up of 24.5 months, 21 patients died, 6 had persistent disease, and 5 achieved complete remission after stem cell transplantation. STAT3 mutation is present in various MNs but not in myeloproliferative neoplasms. It is often an early event or occurs upon leukemic transformation, which suggests an important role in the pathogenesis and progression of MNs by activating the JAK-STAT pathway. It may help determine a subset of patients with MNs who may benefit from targeted therapy. See related commentary by Hochman and Frank, p. 4554.

The STAT3 inhibitor B9 alleviates lipopolysaccharide-induced acute lung injury through its anti-inflammatory effects

The development of acute lung injury (ALI), a common respiratory condition with multiple causes, is significantly influenced by the pro-inflammatory environment of signal transducer and activator of transcription 3 (STAT3) in macrophages. Our study aimed to evaluate the anti-inflammatory effects of B9 (N-(4-hydroxyphenyl)-9, 10-dioxo-9, 10-dihydroanthracene-2-sulfonamide), a novel inhibitor targeting the STAT3 SH2 domain, in macrophages and to assess its therapeutic potential for ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI. We found that B9 (30 mg/kg) significantly reduced lung pathological damage and neutrophil infiltration caused by the intratracheal administration of LPS. Additionally, the high expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in alveolar lavage fluid was also inhibited by B9 treatment. The decreased expression of CD86 and increased CD206 in lung tissue demonstrated the anti-inflammatory effect of B9, which was due to its inhibition of the STAT3 signaling pathway in macrophages of ALI mice. Furthermore, B9 suppressed the activation of RAW264.7 cells induced by LPS, characterized by its ability to inhibit the activation of iNOS and STAT3 in a dose-dependent manner, as well as reduce the secretion of IL-6 and IL-1β. The in vivo preliminary safety evaluation indicated that B9 had a favorable safety profile at the administered doses. These results suggest that B9 exerts a therapeutic effect on LPS-induced ALI, potentially by preventing the phosphorylation of STAT3 Y705 and S727 without affecting the STAT3 protein level. Taken together, these findings provide a foundation for developing B9 as a novel anti-inflammatory agent for ameliorating LPS-induced ALI.

Identifying p56lck SH2 Domain Inhibitors Using Molecular Docking and In Silico Scaffold Hopping

Bacterial infections are the second-leading cause of death, globally. The prevalence of antibacterial resistance has kept the demand strong for the development of new and potent drug candidates. It has been demonstrated that Src protein tyrosine kinases (TKs) play an important role in the regulation of inflammatory responses to tissue injury, which can trigger the onset of several severe diseases. We carried out a search for novel Src protein TK inhibitors, commencing from reported highly potent anti-bacterial compounds obtained using the Mannich reaction, using a combination of e-pharmacophore modeling, virtual screening, ensemble docking, and core hopping. The top-scoring compounds from ligand-based virtual screening were modified using protein structure-based design approaches, and their binding to the Src homology-2 domain of p56lck TK was predicted using ensemble molecular docking. We have prepared a database of 202 small molecules and have identified six novel top hits that can be subjected to further investigation. We have also performed in silico ADMET property prediction for the hit compounds. This combined computer-aided drug design approach can serve as a starting point for identifying novel TK inhibitors that could be further subjected to in vitro studies and validation of antimicrobial activity.

Genome-Wide Identification and Expression Analysis of the STAT Family in Reeve's Turtle (Mauremys reevesii).

The Stat (signal transducer and activator of transcription) gene family plays a vital role in regulating immunity and the processes of cellular proliferation, differentiation, and apoptosis across diverse organisms. Although the functions of Stat genes in immunity have been extensively documented in many mammals, limited data are available for reptiles. We used phylogenetic analysis to identify eight putative members of the Stat family (Stat1-1, Stat1-2, Stat2, Stat3, Stat4, Stat5b, Stat6-1, and Stat6-2) within the genome of M. reevesii, a freshwater turtle found in East Asia. Sequence analysis showed that the Stat genes contain four conserved structural domains protein interaction domain, coiled-coil domain, DNA-binding domain, and Src homology domain 2. In addition, Stat1, Stat2, and Stat6 contain TAZ2bind, Apolipo_F, and TALPID3 structural domains. The mRNA levels of Stat genes were upregulated in spleen tissues at 4, 8, 12, and 16h after administration of lipopolysaccharide, a potent activator of the immune system. Stat5b expression at 12-h LPS post-injection exhibited the most substantial difference from the control. The expression of Stat5b in spleen tissue cellular was verified by immunofluorescence. These results suggest that Stat5b plays a role in the immune response of M. reevesii and may prove to be as a positive marker of an immune response in future studies.

Cymric, a Maternal and Zygotic HTK-16-Like SHARK Family Tyrosine Kinase Gene, Is Disrupted in Molgula occulta, a Tailless Ascidian.

AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, cymric (Uro-1), a HTK-16-like (HydraTyrosineKinase-16) gene, identified in a subtractive screen for maternal ascidian cDNAs in Molgula oculata, an ascidian species with a tadpole larva. The cymric gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed M. oculata but is not detected in eggs or embryos of the closely related tailless species Molgula occulta. There is a large insertion in cymric in the M. occulta genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The cymric amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the HTK-16-like gene found in other ascidians. Thus, the ascidian cymric genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that cymric is lacking the tyrosine kinase domain in the tailless M. occulta, although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic HTK-16-like tyrosine kinase is another described pseudogene from M. occulta and appears not to be necessary for adult development.

Revealing the role of the X25 domains through the characterization of truncated variants of amylopullulanase enzyme from Thermoanaerobacter brockii brockii

To understand the role of the X25 domains of the amylopullulanase enzyme from Thermoanaerobacter brockii brockii (T. brockii brockii), four truncated variants that are TbbApuΔX25-1-SH3 (S130-A1484), TbbApuΔX25-2-SH3 (T235-A1484), TbbApuΔX25-1-CBM20 (S130-P1254), and TbbApuΔX25-2-CBM20 (T235-P1254) were constructed, expressed and characterized together with the SH3 and CBM20 domain truncated variants (TbbApuΔSH3 (V1-A1484) and TbbApuΔCBM20 (V1-P1254). TbbApuΔSH3 showed improved affinity and specificity for both pullulan and soluble starch than full-length TbbApu with lower Km and higher kcat/Km values. It indicates that SH3 is a disposable domain without any effect on the activity and stability of the enzyme. However, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20, TbbApuΔX25-2-CBM20 (T235-P1254) and TbbApuΔCBM20 showed higher Km and lower kcat/Km values than TbbApuΔSH3 to both soluble starch and pullulan. It specifies that the X25 domains and CBM20 play an important role in both α-amylase and pullulanase activity. Also, it is revealed that while truncation of the CBM20 domain as starch binding domain (SBD) did not affect on raw starch binding ability of the enzyme, truncation of both X25 domains caused almost complete loss of the raw starch binding ability of the enzyme. All these results enlightened the function of the X25 domains that play a more crucial role than CBM20 in the enzyme's binding to raw starch and also play a crucial role in its activity.

Determination of promising inhibitors for N-SH2 domain of SHP2 tyrosine phosphatase: an in silico study.

There are many genes that produce proteins related to diseases and these proteins can be targeted with drugs as a potential therapeutic approach. Recent advancement in drug discovery techniques have created new opportunities for treating variety of diseases by targeting disease-related proteins. Structure-based drug discovery is a faster and more cost-effective approach than traditional methods. SHP2 phosphatase, encoded by the PTPN11 gene, has been the focus of much attention due to its involvement in many types of diseases. The biological function of SHP2 is enabled mostly by protein-protein interaction through its SH2 domains. In this study, we report the identification of a potential small molecule inhibitor for the N-SH2 domain of SHP2 by structure-based drug discovery approach. We utilized molecular docking studies, followed by molecular dynamics simulations and MM/PBSA calculations, to analyze compounds retrieved from the Broad's Drug Repurposing Hub and ZINC15 databases. We selected 10 hit compounds with the best docking scores from the libraries and examined their binding properties in the N-SH2 domain. We found that compound CID 60838 (Irinotecan) was the most suitable compound with a binding free energy value of - 64.45kcal/mol and significant interactions with the target residues in the domain.

Direct Interaction of Minocycline to p47phox Contributes to its Attenuation of TNF-α-Mediated Neuronal PC12 Cell Death: Experimental and Simulation Validation.

Minocycline, a repurposed approved medication, shows promise in treating neurodegeneration. However, the specific pathways targeted by minocycline remain unclear despite the identification of molecular targets. This study explores minocycline's potential protective effects against TNF-α-mediated neuronal death in PC12 cells, with a focus on unraveling its interactions with key molecular targets. The study begins by exploring minocycline's protective role against TNF-α-mediated neuronal death in PC12 cells, showcasing a substantial reduction in cleaved caspase-3 expression, DNA fragmentation, and intracellular ROS levels following minocycline pretreatment. Subsequently, a comprehensive analysis utilizing pull-down assays, computational docking, mutation analysis, molecular dynamics simulations, and free energy calculations is conducted to elucidate the direct interaction between minocycline and p47phox-the organizer subunit of NADPH oxidase-2 (NOX2) complex. Computational insights, including a literature survey and analysis of key amino acid residues, reveal a potential binding site for minocycline around Trp193 and Cys196. In silico substitutions of Trp193 and Cys196 further confirm their importance in binding with minocycline. These integrated findings underscore minocycline's protective mechanisms, linking its direct interaction with p47phox to the modulation of NOX2 activity and attenuation of NOX-derived ROS generation. Minocycline demonstrates protective effects against TNF-α-induced PC12 cell death, potentially linked to its direct interaction with p47phox. This interaction leads to a reduction in NOX2 complex assembly, ultimately attenuating NOX-derived ROS generation. These findings hold significance for researchers exploring neuroprotection and the development of p47phox inhibitors.

ZNF692 promotes the migration and response to immunotherapy of clear cell renal cell carcinoma cells by targeting metabolic pathway

Clear cell renal cell carcinoma (ccRCC), with high mortality and poor prognosis, is the most common type of renal malignancy. It is necessary to identify new biomarkers that can serve as indicators for the detection of ccRCC at its early stages. In this study, we analyzed the role of classical zinc finger protein 692 (ZNF692) in ccRCC using datasets from The Cancer Genome Atlas (TCGA) and Single Cell Portal and immunohistochemical (IHC) staining of a tissue-microarray, and analyzed the function of ZNF692 in ccRCC cells. The analyses indicated that ZNF692 was upregulated in ccRCC samples compared with normal or paracancerous control samples (P < 0.001) and that the expression of this gene was linked to poor overall survival (HR = 2.1, P < 0.0001). The knockdown of ZNF692 inhibited the proliferation and migration of ccRCC cells by target GTPase-activating protein (SH3 domain)-binding protein 2 (G3BP2), and transmembrane 9 superfamily member 2 (TM9SF2)). T, B, proximal, and collecting tubule cells are the dominant cell types in normal kidney tissue where ZNF692 is expressed. In addition, immune checkpoint blockade (ICB) therapy dramatically changed the expression patterns of ZNF692. Collectively, these data indicate that ZNF692 may serve as prognosis, and as a potential indicator of the response to ICB therapy, a possibility needs to be verified by a case‒control study.

STAT5B SH2 variants disrupt mammary enhancers and the stability of genetic programs during pregnancy.

During pregnancy, mammary tissue undergoes expansion and differentiation, leading to lactation, a process regulated by the hormone prolactin through the JAK2-STAT5 pathway. STAT5 activation is key to successful lactation making the mammary gland an ideal experimental system to investigate the impact of human missense mutations on mammary tissue homeostasis. Here, we investigated the effects of two human variants in the STAT5B SH2 domain, which convert tyrosine 665 to either phenylalanine (Y665F) or histidine (Y665H), both shown to activate STAT5B in cell culture. We ported these mutations into the mouse genome and found distinct and divergent functions. Homozygous Stat5bY665H mice failed to form functional mammary tissue, leading to lactation failure, with impaired alveolar development and greatly reduced expression of key differentiation genes. STAT5BY665H failed to recognize mammary enhancers and impeded STAT5A binding. In contrast, mice carrying the Stat5bY665F mutation exhibited abnormal precocious development, accompanied by an early activation of the mammary transcription program and the induction of otherwise silent genetic programs. Physiological adaptation was observed in Stat5bY665H mice as continued exposure to pregnancy hormones led to lactation. In summary, our findings highlight that human STAT5B variants can modulate their response to cytokines and thereby impact mammary homeostasis and lactation.

LncRNA PAARH impacts liver cancer cell proliferation by engaging miR‑6512‑3p to target LASP1.

Long non-coding (lnc)RNAs serve a pivotal role as regulatory factors in carcinogenesis. The present study aimed to assess the involvement of the lncRNA progression and angiogenesis-associated RNA in hepatocellular carcinoma (PAARH) in liver cancer, along with the associated underlying mechanism. Through the use of reverse transcription-quantitative (RT-q)PCR, differences in the expression levels of PAARH in HepG2, HEP3B2.1.7, HCCLM3, Huh-7 and MHCC97-H liver cancer cell lines and THLE-2 epithelial cell lines were evaluated. The liver cancer cell line with the greatest, significantly different, level of expression relative to the normal liver cell line was selected for subsequent experiments. Using ENCORI database, the putative target genes of the microRNA (miR) miR-6512-3p were predicted. Cells were then transfected with lentiviruses carrying short-hairpin-PAARH to interfere with PAARH expression. Subsequently, HepG2 liver cancer cells were transfected with a miR-6512-3p mimic and an inhibitor, and the expression levels of miR-6512-3p and the LIM and SH3 domain protein 1 (LASP1) in cells were assessed using RT-qPCR analysis. Cell proliferation was subsequently evaluated using colony formation assays, and immunofluorescence and western blotting were used to assess the expression level of LASP1 in transfected cells. The binding interaction between miR-6512-3p and LASP1 was further evaluated using a dual-luciferase reporter gene assay. Liver cancer cells were found to exhibit higher expression levels of PAARH compared with normal liver cells. Following PAARH interference, the expression level of miR-6512-3p was significantly increased, whereas that of LASP1 was significantly decreased, resulting in a reduction in cell proliferation. In liver cancer cells, miR-6512-3p overexpression led to a significant reduction in the LASP1 level and reduced proliferation, whereas suppressing miR-6512-3p led to a significant increase in LASP1 levels and increased proliferation. Additionally, the inhibition of miR-6512-3p caused the states of low LASP1 expression and reduced cell proliferation to be reversed. LASP1, a recently identified target gene of miR-6512-3p, was demonstrated to be suppressed by miR-6512-3p overexpression, thereby inhibiting liver cancer cell proliferation. Taken together, the findings of the present study demonstrate that the lncRNA PAARH may enhance liver cancer cell proliferation by engaging miR-6512-3p to target LASP1.

Understanding the Energy Landscape of Intrinsically Disordered Protein Ensembles.

A substantial portion of various organisms' proteomes comprises intrinsically disordered proteins (IDPs) that lack a defined three-dimensional structure. These IDPs exhibit a diverse array of conformations, displaying remarkable spatiotemporal heterogeneity and exceptional conformational flexibility. Characterizing the structure or structural ensemble of IDPs presents significant conceptual and methodological challenges owing to the absence of a well-defined native structure. While databases such as the Protein Ensemble Database (PED) provide IDP ensembles obtained through a combination of experimental data and molecular modeling, the absence of reaction coordinates poses challenges in comprehensively understanding pertinent aspects of the system. In this study, we leverage the energy landscape visualization method (JCTC, 6482, 2019) to scrutinize four IDP ensembles sourced from PED. ELViM, a methodology that circumvents the need for a priori reaction coordinates, aids in analyzing the ensembles. The specific IDP ensembles investigated are as follows: two fragments of nucleoporin (NUL: 884-993 and NUS: 1313-1390), yeast sic 1 N-terminal (1-90), and the N-terminal SH3 domain of Drk (1-59). Utilizing ELViM enables the comprehensive validation of ensembles, facilitating the detection of potential inconsistencies in the sampling process. Additionally, it allows for identifying and characterizing the most prevalent conformations within an ensemble. Moreover, ELViM facilitates the comparative analysis of ensembles obtained under diverse conditions, thereby providing a powerful tool for investigating the functional mechanisms of IDPs.