Search Results (24)

Numerous studies have documented the involvement of p130Cas (Crk-associated substrate) in a wide range of cellular processes across different types of cells. These processes encompass cell transformation, the connection between the extracellular matrix and the actin cytoskeleton, cell migration and invasion, and cardiovascular development. Moreover, p130Cas has been associated with the regulation of various physiological processes, including mammary, bone, brain, muscle, and liver homeostasis. The diverse functions of p130Cas can be attributed to its possession of multiple protein–protein interaction domains, which sets it apart as a unique class of adaptor protein. It is well established that p130Cas interacts critically with the CT10 regulator of kinase (Crk) adaptor protein family members, including CrkII, CrkI, and Crk-like (CrkL), which is the basis for the naming of the Cas family. The Crk family proteins play a crucial role in integrating signals from various sources, such as growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. An increasing body of evidence suggests that the dysregulation of Crk family proteins is linked to various human diseases, including cancer and increased susceptibility to pathogen infections. This review focuses primarily on the structural and functional aspects of the interaction between p130Cas and the Crk family proteins, providing insights into how these proteins regulate specific signaling events. Furthermore, we delve into the functions of p130Cas and the Crk family proteins in both normal and tumor cells to gain a comprehensive understanding of their collaborative roles in cellular physiology and pathology. This review demonstrates that tumor cell migration and invasion are the two cellular functions that have been studied the most for the p130Cas-Crk/CrkL axis. Understanding the tumor cell migration and invasion that require both p130Cas and Crk/CrkL is necessary to further evaluate the role of the p130Cas-Crk/CrkL axis in cancer. Establishing the contribution of the p130Cas-Crk/CrkL axis to cancer will facilitate the development of cancer drugs targeting the axis to inhibit cancer cell dissemination and improve patient outcomes. Full article

22q11.2 is a region prone to chromosomal rearrangements due to the presence of eight large blocks of low-copy repeats (LCR22s). The 3 Mb 22q11.2 “typical deletion”, between LCR22-A and D, causes a fairly well-known clinical picture, while the effects of smaller CNVs harbored in this interval are still to be fully elucidated. Nested deletions, flanked by LCR22B-D, LCR22B-C, or LCR22C-D, are very rare and are collectively described as “central deletions”. The LCR22C-D deletion (CDdel) has never been separately analyzed. In this paper, we focused only on CDdel, evaluating its gene content and reviewing the literature and public databases in order to obtain new insights for the classification of this CNV. At first glance, CDdels are associated with a broad phenotypic spectrum, ranging from clinically normal to quite severe phenotypes. However, the frequency of specific clinical traits highlights that renal/urinary tract abnormalities, cardiac defects, and neurological/behavioral disorders are much more common in CDdel than in the general population. This frequency is too high to be fortuitous, indicating that CDdel is a predisposing factor for these phenotypic traits. Among the genes present in this interval, CRKL is an excellent candidate for cardiac and renal defects. Even if further data are necessary to confirm the role of CDdels, according to our review, this CNV fits into the class of ‘likely pathogenic’ CNVs. Full article

The Hippo pathway transducers yes-associated protein (YAP) and WW-domain containing transcription regulator 1 (WWTR1/TAZ) are key regulators of liver tumorigenesis, promoting tumor formation and progression. Although the first inhibitors are in clinical trials, targeting the relevant upstream regulators of YAP/TAZ activity could prove equally beneficial. To identify regulators of YAP/TAZ activity in hepatocarcinoma (HCC) cells, we carried out a proximity labelling approach (BioID) coupled with mass spectrometry. We verified CRK-like proto-oncogene adaptor protein (CRKL) as a new YAP-exclusive interaction partner. CRKL is highly expressed in HCC patients, and its expression is associated with YAP activity as well as poor survival prognosis. In vitro experiments demonstrated CRKL-dependent cell survival and the loss of YAP binding induced through actin disruption. Moreover, we delineated the activation of the JNK/JUN pathway by CRKL, which promoted YAP transcription. Our data illustrate that CRKL not only promoted YAP activity through its binding but also through the induction of YAP transcription by JNK/JUN activation. This emphasizes the potential use of targeting the JNK/JUN pathway to suppress YAP expression in HCC patients. Full article

Male and female Chinese soft-shelled turtles (Pelodiscus sinensis) have sex-dimorphic growth patterns, and males have higher commercial value because of their larger size and thicker calipash. Thus, developing sex-specific markers is beneficial to studies on all-male breeding in P. sinensis. Here, we developed an accurate and efficient workflow for the screening of sex-specific sequences with ZW or XY sex determination systems. Based on this workflow, female and male P. sinensis reference genomes of 2.23 Gb and 2.26 Gb were obtained using de novo assembly. After aligning and filtering, 4.01 Mb female-specific sequences were finally identified. Subsequently, the seven developed sex-specific primer pairs were 100% accurate in preliminary, population, and embryonic validation. The presence and absence of bands for the primers of P44, P45, P66, P67, P68, and P69, as well as two and one bands for the PB1 primer, indicate that the embryos are genetically female and male, respectively. NR and functional annotations identified several sex-determining candidate genes and related pathways, including Ran, Eif4et, and Crkl genes, and the insulin signaling pathway and the cAMP signaling pathway, respectively. Collectively, our results reveal that a ZW-type sex-determination system is present in P. sinensis and provide novel insights for the screening of sex-specific markers, sex-control breeding, and the studies of the sex determination mechanism of P. sinensis. Full article

Mutations in the GBA1 gene increase the risk of developing Parkinson’s disease (PD). However, most carriers of GBA1 mutations do not develop PD throughout their lives. The mechanisms of how GBA1 mutations contribute to PD pathogenesis remain unclear. Cerebrospinal fluid (CSF) is used for detecting pathological conditions of diseases, providing insights into the molecular mechanisms underlying neurodegenerative disorders. In this study, we utilized the proximity extension assay to examine the levels of metabolism-linked protein in the CSF from 17 PD patients carrying GBA1 mutations (GBA1-PD) and 17 idiopathic PD (iPD). The analysis of CSF secretome in GBA1-PD identified 11 significantly altered proteins, namely FKBP4, THOP1, GLRX, TXNDC5, GAL, SEMA3F, CRKL, APLP1, LRP11, CD164, and NPTXR. To investigate GBA1-associated CSF changes attributed to specific neuronal subtypes responsible for PD, we analyzed the cell culture supernatant from GBA1-PD-induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic (mDA) neurons. The secretome analysis of GBA1-PD iPSC-derived mDA neurons revealed that five differently regulated proteins overlapped with those identified in the CSF analysis: FKBP4, THOP1, GLRX, GAL, and CRKL. Reduced intracellular level of the top hit, FKPB4, was confirmed via Western Blot. In conclusion, our findings identify significantly altered CSF GBA1-PD-associated proteins with FKPB4 being firmly attributed to mDA neurons. Full article

The steel tube-reinforced concrete (STRC) shear wall plays an important role in the seismic design of high-rise building structures. Due to the synergistic collaboration between steel tubes and concrete, they effectively enhance the ductility and energy dissipation capacity of conventional shear walls. To identify vulnerable areas prone to brittle failure and optimize the design, it is essential to develop a rapid method for identifying the failure mode of STRC shear walls. In this study, a fast identification method of STCR shear wall failure modes based on a Blending fusion model with Generative Adversarial Network (GAN) augmented data is proposed. The GAN is employed to address the issue of inadequate experimental data by generating new samples. This method combines classification boosting (Catboost), Random Forest (RF), K-Nearest Neighbors (KNN), and Least Absolute Shrinkage and Selection Operator (LASSO) to establish the Blending-CRKL fusion model to improve the prediction accuracy of the failure mode of STRC shear walls. The results reveal a significant improvement in the prediction performance of KNN, Backpropagation Neural Network (BPNN), RF, Light Gradient Boosting Machine (LightGBM), Catboost, and Blending-CRKL models after augmenting the training set with GAN. On average, the accuracy increased by 13%, precision increased by 81%, recall increased by 48%, and F1 score increased by 67%. The proposed Blending-CRKL fusion model outperforms the tested KNN, BPNN, RF, LightGBM, and Catboost models, achieving an accuracy rate of 97% in predicting the failure mode of STRC shear walls. Additionally, the stability and robustness of the Blending-CRKL model were validated, while the important features and value ranges of different failure modes were analyzed. This study provides a reference for the rapid identification of the failure mode of STRC shear walls. Full article

Breast cancer (BC) ranks in the top five malignant tumors in terms of morbidity and mortality rates. Among BC subtypes, TNBC has a high recurrence rate and metastasis rate and the worst prognosis. However, the exact mechanism by which TNBC develops is unclear. Here, we show that the deubiquitinase USP53 contributes to tumor growth and metastasis in TNBC. USP53 is overexpressed in TNBC, and this phenotype is linked to a poor prognosis. Functionally, USP53 promotes TNBC cell proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT). More importantly, USP53 decreases the chemosensitivity of BC cells by enhancing v-crk sarcoma virus CT10 oncogene homologue (avian)-like (CRKL) expression. Mechanistically, USP53 directly binds CRKL to stabilize and deubiquitinate it, thereby preventing CRKL degradation. Overall, we discovered that USP53 deubiquitinates CRKL, encourages tumor development and metastasis, and reduces chemosensitivity in TNBC. These findings imply that USP53 might represent a new therapeutic target for the treatment of TNBC. Full article

The IRS (insulin receptor substrate) family of scaffold proteins includes insulin receptor substrate-4 (IRS4), which is expressed only in a few cell lines, including human kidney, brain, liver, and thymus and some cell lines. Its N-terminus carries a phosphotyrosine-binding (PTB) domain and a pleckstrin homology domain (PH), which distinguishes it as a member of this family. In this paper, we collected data about the molecular mechanisms that explain the relevance of IRS4 in the development of cancer and identify IRS4 differences that distinguish it from IRS1 and IRS2. Search engines and different databases, such as PubMed, UniProt, ENSEMBL and SCANSITE 4.0, were used. We used the name of the protein that it encodes “(IRS-4 or IRS4)”, or the combination of these terms with the word “(cancer)” or “(human)”, for searches. Terms related to specific tumor pathologies (“breast”, “ovary”, “colon”, “lung”, “lymphoma”, etc.) were also used. Despite the lack of knowledge on IRS4, it has been reported that some cancers and benign tumors are characterized by high levels of IRS-4 expression. Specifically, the role of IRS-4 in different types of digestive tract neoplasms, gynecological tumors, lung cancers, melanomas, hematological tumors, and other less common types of cancers has been shown. IRS4 differs from IRS1 and IRS2 in that can activate several oncogenes that regulate the PI3K/Akt cascade, such as BRK and FER, which are characterized by tyrosine kinase-like activity without regulation via extracellular ligands. In addition, IRS4 can activate the CRKL oncogene, which is an adapter protein that regulates the MAP kinase cascade. Knowledge of the role played by IRS4 in cancers at the molecular level, specifically as a platform for oncogenes, may enable the identification and validation of new therapeutic targets. Full article

The S2 subunit serves a crucial role in infectious bronchitis virus (IBV) infection, particularly in facilitating membrane fusion. Using reverse genetic techniques, mutant strains of the S2 locus exhibited substantially different syncytium-forming abilities in chick embryonic kidney cells. To determine the precise formation mechanism of syncytium, we demonstrated the co-ordinated role of Abl2 and its mediated cytoskeletal regulatory pathway within the S2 subunit. Using a combination of fluorescence quantification, RNA silencing, and protein profiling techniques, the functional role of S2 subunits in IBV-infected cells was exhaustively determined. Our findings imply that Abl2 is not the primary cytoskeletal regulator, the viral S2 component is involved in indirect regulation, and the three different viral strains activate various cytoskeletal regulatory pathways through Abl2. CRK, CRKL, ABI1, NCKAP1, and ENAH also play a role in cytoskeleton regulation. Our research provides a point of reference for the development of an intracellular regulatory network for the S2 subunit and a foundation for the rational design of antiviral drug targets against Abl2. Full article

Skeletal muscle is an important economic trait in duck breeding; however, little is known about the molecular mechanisms of its embryonic development. Here, the transcriptomes and metabolomes of breast muscle of Pekin duck from 15 (E15_BM), 21 (E21_BM), and 27 (E27_BM) days of incubation were compared and analyzed. The metabolome results showed that the differentially accumulated metabolites (DAMs), including the up-regulated metabolites, l-glutamic acid, n-acetyl-1-aspartylglutamic acid, l-2-aminoadipic acid, 3-hydroxybutyric acid, bilirubin, and the significantly down-regulated metabolites, palmitic acid, 4-guanidinobutanoate, myristic acid, 3-dehydroxycarnitine, and s-adenosylmethioninamine, were mainly enriched in metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of cofactors, protein digestion and absorption, and histidine metabolism, suggesting that these pathways may play important roles in the muscle development of duck during the embryonic stage. Moreover, a total of 2142 (1552 up-regulated and 590 down-regulated), 4873 (3810 up-regulated and 1063 down-regulated), and 2401 (1606 up-regulated and 795 down-regulated) DEGs were identified from E15_BM vs. E21_BM, E15_BM vs. E27_BM and E21_BM vs. E27_BM in the transcriptome, respectively. The significantly enriched GO terms from biological processes were positive regulation of cell proliferation, regulation of cell cycle, actin filament organization, and regulation of actin cytoskeleton organization, which were associated with muscle or cell growth and development. Seven significant pathways, highly enriched by FYN, PTK2, PXN, CRK, CRKL, PAK, RHOA, ROCK, INSR, PDPK1, and ARHGEF, were focal adhesion, regulation of actin cytoskeleton, wnt signaling pathway, insulin signaling pathway, extracellular matrix (ECM)-receptor interaction, cell cycle, and adherens junction, which participated in regulating the development of skeletal muscle in Pekin duck during the embryonic stage. KEGG pathway analysis of the integrated transcriptome and metabolome indicated that the pathways, including arginine and proline metabolism, protein digestion and absorption, and histidine metabolism, were involved in regulating skeletal muscle development in embryonic Pekin duck. These findings suggested that the candidate genes and metabolites involved in crucial biological pathways may regulate muscle development in the Pekin duck at the embryonic stage, and increased our understanding of the molecular mechanisms underlying the avian muscle development. Full article

We have previously established that the integrity of the induced blood–brain barrier (iBBB) formed by brain microvascular endothelial cells derived from the iPSC of 22q11.2 DS (22q11.2 Deletion Syndrome, also called DiGeorge Syndrome) patients is compromised. We tested the possibility that the haploinsufficiency of CRKL, a gene within the 22q11.2 DS deletion region, contributes to the deficit. The CRKL is a major substrate of the Abl tyrosine kinase, and the Abl/CRKL signaling pathway is critical for endothelial barrier functions. Imatinib, an FDA-approved drug, inhibits Abl kinase and has been used to treat various disorders involving vascular leakages. To test if imatinib can restore the compromised iBBB, we treated the patient’s iBBB with imatinib. After treatment, both trans-endothelial electrical resistance and solute permeability returned to comparable levels of the control iBBB. Correspondingly, changes in tight junctions and endothelial glycocalyx of the iBBB were also restored. Western blotting showed that imatinib increased the level of active forms of the CRKL protein. A transcriptome study revealed that imatinib up-regulated genes in the signaling pathways responsible for the protein modification process and down-regulated those for cell cycling. The KEGG pathway analysis further suggested that imatinib improved the gene expression of the CRKL signaling pathway and tight junctions, which agrees with our expectations and the observations at protein levels. Our results indicate that the 22q11.2DS iBBB is at least partially caused by the haploinsufficiency of CRKL, which can be rescued by imatinib via its effects on the Abl/CRKL signaling pathway. Our findings uncover a novel disease mechanism associated with 22q11.2DS. Full article

Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only Aeromonas salmonicida exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins. Structural modeling of AexT indicated a target binding site that shared homology with Pseudomonas aeruginosa Exoenzyme T (ExoT) but not with Exoenzyme S (ExoS). Biochemical analyses confirmed that the catalytic activities of both exotoxins were stimulated by agmatine, indicating that they ADP-ribosylate arginine residues in their targets. Side-by-side comparison of target protein modification showed that AexT had activity toward the SH2 domain of the Crk-like protein (CRKL), a known target for ExoT. We found that both AexT and ExoT ADP-ribosylated actin and in both cases, the modification compromised actin polymerization. Our results indicate that AexT and ExoT are functional homologs that affect cytoskeletal integrity via actin and signaling pathways to the cytoskeleton. Full article

Ph-like subtypes with CRLF2 abnormalities are frequent among Hispano–Latino children with pre-B ALL. Therefore, there is solid ground to suggest that this subtype is frequent in Mexican patients. The genomic complexity of Ph-like subtype constitutes a challenge for diagnosis, as it requires diverse genomic methodologies that are not widely available in diagnostic centers in Mexico. Here, we propose a diagnostic strategy for Ph-like ALL in accordance with our local capacity. Pre-B ALL patients without recurrent gene fusions (104) were classified using a gene-expression profile based on Ph-like signature genes analyzed by qRT-PCR. The expressions of the CRLF2 transcript and protein were determined by qRT-PCR and flow cytometry. The P2RY8::CRLF2, IGH::CRLF2, ABL1/2 rearrangements, and Ik6 isoform were screened using RT-PCR and FISH. Surrogate markers of Jak2-Stat5/Abl/Ras pathways were analyzed by phosphoflow. Mutations in relevant kinases/transcription factors genes in Ph-like were assessed by target-specific NGS. A total of 40 patients (38.5%) were classified as Ph-like; of these, 36 had abnormalities associated with Jak2-Stat5 and 4 had Abl. The rearrangements IGH::CRLF2,P2RY8::CRLF2, and iAMP21 were particularly frequent. We propose a strategy for the detection of Ph-like patients, by analyzing the overexpression/genetic lesions of CRLF2, the Abl phosphorylation of surrogate markers confirmed by gene rearrangements, and Sanger sequencing. Full article

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains. Full article

Crkl is a protein involved in the onset of several cancer pathologies that exerts its function only through its protein–protein interaction domains, a SH2 domain and two SH3 domains. SH3 domains are small protein interaction modules that mediate the binding and recognition of proline-rich sequences. One of the main physiological interactors of Crkl is C3G (also known as RAPGEF1), an interaction with key implications in regulating cellular growth and differentiation, cell morphogenesis and adhesion processes. Thus, understanding the interaction between Crkl and C3G is fundamental to gaining information about the molecular determinants of the several cancer pathologies in which these proteins are involved. In this paper, through a combination of fast kinetics at different experimental conditions and site-directed mutagenesis, we characterize the binding reaction between the N-SH3 domain of Crkl and a peptide mimicking a specific portion of C3G. Our results show a clear effect of pH on the stability of the complex, due to the protonation of negatively charged residues in the binding pocket of N-SH3. Our results are discussed under the light of previous work on SH3 domains. Full article