BACKGROUND: Programmed cell death is an important mechanism for the development of hepatic ischemia and reperfusion (IR) injury, and multiple novel forms of programmed cell death are involved in the pathological process of hepatic IR. ERRFI1 is involved in the regulation of cell apoptosis in myocardial IR. However, the function of ERRFI1 in hepatic IR injury and its modulation of programmed cell death remain largely unknown.
METHODS: Here, we performed functional and molecular mechanism studies in hepatocyte-specific knockout mice and ERRFI1-silenced hepatocytes to investigate the significance of ERRFI1 in hepatic IR injury. The histological severity of livers, enzyme activities, hepatocyte apoptosis and ferroptosis were determined.
RESULTS: ERRFI1 expression increased in liver tissues from mice with IR injury and hepatocytes under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions. Hepatocyte-specific ERRFI1 knockout alleviated IR-induced liver injury in mice by reducing cell apoptosis and ferroptosis. ERRFI1 knockdown reduced apoptotic and ferroptotic hepatocytes induced by OGD/R. Mechanistically, ERRFI1 interacted with GRB2 to maintain its stability by hindering its proteasomal degradation. Overexpression of GRB2 abrogated the effects of ERRFI1 silencing on hepatocyte apoptosis and ferroptosis.
CONCLUSIONS: Our results revealed that the ERRFI1-GRB2 interaction and GRB2 stability are essential for ERRFI1-regulated hepatic IR injury, indicating that inhibition of ERRFI1 or blockade of the ERRFI1-GRB2 interaction may be potential therapeutic strategies in response to hepatic IR injury.

The phosphoinositide-3 kinase (PI3K), a heterodimeric enzyme, plays a pivotal role in cellular metabolism and survival. Its deregulation is associated with major human diseases, particularly cancer. The p85 regulatory subunit of PI3K binds to the catalytic p110 subunit via its C-terminal domains, stabilising it in an inhibited state. Certain Src homology 3 (SH3) domains can activate p110 by binding to the proline-rich (PR) 1 motif located at the N-terminus of p85. However, the mechanism by which this N-terminal interaction activates the C-terminally bound p110 remains elusive. Moreover, the intrinsically poor ligand selectivity of SH3 domains raises the question of how they can control PI3K. Combining structural, biophysical, and functional methods, we demonstrate that the answers to both these unknown issues are linked: PI3K-activating SH3 domains engage in additional \"tertiary\" interactions with the C-terminal domains of p85, thereby relieving their inhibition of p110. SH3 domains lacking these tertiary interactions may still bind to p85 but cannot activate PI3K. Thus, p85 uses a functional selection mechanism that precludes nonspecific activation rather than nonspecific binding. This separation of binding and activation may provide a general mechanism for how biological activities can be controlled by promiscuous protein-protein interaction domains.

Growth-factor-receptor-binding protein 2 (GRB2) is a non-enzymatic adaptor protein that plays a pivotal role in precisely regulated signaling cascades from cell surface receptors to cellular responses, including signaling transduction and gene expression. GRB2 binds to numerous target molecules, thereby modulating a complex cell signaling network with diverse functions. The structural characteristics of GRB2 are essential for its functionality, as its multiple domains and interaction mechanisms underpin its role in cellular biology. The typical signaling pathway involving GRB2 is initiated by the ligand stimulation to its receptor tyrosine kinases (RTKs). The activation of RTKs leads to the recruitment of GRB2 through its SH2 domain to the phosphorylated tyrosine residues on the receptor. GRB2, in turn, binds to the Son of Sevenless (SOS) protein through its SH3 domain. This binding facilitates the activation of Ras, a small GTPase, which triggers a cascade of downstream signaling events, ultimately leading to cell proliferation, survival, and differentiation. Further research and exploration into the structure and function of GRB2 hold great potential for providing novel insights and strategies to enhance medical approaches for related diseases. In this review, we provide an outline of the proteins that engage with domains of GRB2, along with the function of different GRB2 domains in governing cellular signaling pathways. This furnishes essential points of current studies for the forthcoming advancement of therapeutic medications aimed at GRB2.

Wilm\'s tumor 1-associating protein (WTAP), a regulatory protein of the m6A methyltransferase complex, has been found to play a role in regulating various physiological and pathological processes. However, the in vivo role of WTAP in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. In this study, we have elucidated the crucial role of WTAP in HCC progression and shown that hepatic deletion of Wtap promotes HCC pathogenesis through activation of multiple signaling pathways. A single dose of diethylnitrosamine injection causes more and larger HCCs in hepatocyte-specific Wtap knockout (Wtap-HKO) mice than Wtapflox/flox mice fed with either normal chow diet or a high-fat diet. Elevated CD36, IGFBP1 (insulin-like growth factor-binding protein 1), and chemokine (C-C motif) ligand 2 (CCL2) expression leads to steatosis and inflammation in the Wtap-HKO livers. The hepatocyte proliferation is dramatically increased in Wtap-HKO mice, which is due to higher activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-3 signaling pathways. Hepatic deletion of Wtap activates the ERK signaling pathway by increasing the protein stability of GRB2 and ERK1/2, which is due to the decreased expression of proteasome-related genes. Restoring PSMB4 or PSMB6 (two key components of the proteasome) leads to the downregulation of GRB2 and ERK1/2 in Wtap-HKO hepatocytes. Mechanistically, WTAP interacts with RNA polymerase II and H3K9ac to maintain expression of proteasome-related genes. These results demonstrate that hepatic deletion of Wtap promotes HCC progression through activating GRB2-ERK1/2-mediated signaling pathway depending on the downregulation of proteasome-related genes especially Psmb4 and Psmb6.

BACKGROUND: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune system dysfunction, along with tissue fibrosis. Our previous study found GRB2 was downregulated by salvianolic acid B, a small molecule drug that attenuated skin fibrosis of SSc.
OBJECTIVE: Here we aim to investigate the role of GRB2 in SSc.
METHODS: The microarray data of SSc skin biopsies in Caucasians were obtained from the Gene Expression Omnibus (GEO) database. The expression of GRB2 was further detected in Chinese SSc and healthy controls. Bleomycin (BLM)-induced skin fibrosis mice were used to explore how GRB2 downregulation affected fibrosis. The apoptosis of EA.hy926 endothelial cells was induced by H2O2 and apoptosis ratio was measured by flow cytometric. Transcriptome and phosphoproteomic analyses were performed to explore the regulated pathway.
RESULTS: The expression of GRB2 was significantly enhanced in SSc patient skin, 1.51-fold in Caucasians and 1.40-fold in Chinese. Double immunofluorescence staining showed the endothelial cells of SSc patient\'s skin highly expressed GRB2. The in vivo study revealed that GRB2 knockdown alleviated skin fibrosis and apoptosis of endothelial cells in BLM mouse skin. The in vitro study showed that GRB2 downregulation inhibited the apoptosis of EA.hy926 and protected them from H2O2-induced hyperpermeability. Moreover, transcriptome and phosphoproteomic analysis suggested the focal adhesion pathway was enriched in GRB2 siRNA transfected endothelial cells.
CONCLUSIONS: Our results demonstrated GRB2 highly expressed in endothelial cells of SSc skin, and inhibiting GRB2 could effectively attenuate BLM-induced skin fibrosis and endothelial cell apoptosis. GRB2 is expected to be a new therapeutic target for SSc.

The role of the membrane-associated RING-CH (MARCH) family in carcinogenesis has been widely studied, but the member of this family, RNF173, has not yet been thoroughly explored in the context of hepatocellular carcinoma (HCC).
With the use of an HCC tissue microarray and IHC staining, we aim to determine the differential expression of RNF173 in HCC patients and its clinical significance. The biological role of RNF173 is investigated through in vitro and in vivo experiments. RNA sequencing, mass spectrometry, and immunoprecipitation are performed to uncover the underlying mechanism of RNF173\'s impact on the development of HCC.
The mRNA and protein levels of RNF173 were significantly lower in HCC tissues than in normal tissues. HCC patients with low RNF173 expression had shorter overall survival and recurrence-free survival, and RNF173 was significantly correlated with tumor number, tumor capsule, tumor differentiation, and BCLC stage. In addition, in vitro and in vivo experiments showed that RNF173 downregulation exacerbated tumor progression, including migration, invasion, and proliferation. GRB2 is a key molecule in the RAF/MEK/ERK pathway. RNF173 inhibits the RAF/MEK/ERK signaling by ubiquitinating and degrading GRB2, thereby suppressing HCC cell proliferation, invasion and migration. Combining clinical samples, we found that HCC patients with high RNF173 and low GRB2 expression had the best prognosis.
RNF173 inhibits the invasion and metastasis of HCC by ubiquitinating and degrading GRB2, thereby suppressing the RAF/MEK/ERK signaling pathway. RNF173 is an independent risk factor for the survival and recurrence of HCC patients. RNF173 may serve as a novel prognostic molecule and potential therapeutic target for HCC. Video Abstract Graphical abstract Model of RNF173 on RAF/MEK/ERK signaling. RNF173 knockdown resulted in impaired ubiquitination and degradation of GRB2, leading to the activation of the RAF/MEK/ERK signaling pathway and promotion of invasion and metastasis in HCC cells.

The basal chordate amphioxus is a model for tracing the origin and evolution of vertebrate immunity. To explore the evolution of immunoreceptor signaling pathways, we searched the associated receptors of the amphioxus Branchiostoma belcheri (Bb) homolog of immunoreceptor signaling adaptor protein Grb2. Mass-spectrum analysis of BbGrb2 immunoprecipitates from B. belcheri intestine lysates revealed a folate receptor (FR) domain- and leucine-rich repeat (LRR)-containing protein (FrLRR). Sequence and structural analysis showed that FrLRR is a membrane protein with a predicted curved solenoid structure. The N-terminal Fr domain contains very few folate-binding sites; the following LRR region is a Slit2-type LRR, and a GPI-anchored site was predicted at the C-terminus. RT-PCR analysis showed FrLRR is a transcription-mediated fusion gene of BbFR-like and BbSlit2-N-like genes. Genomic DNA structure analysis implied the B. belcheri FrLRR gene locus and the corresponding locus in Branchiostoma floridae might be generated by exon shuffling of a Slit2-N-like gene into an FR gene. RT-qPCR, immunostaining, and immunoblot results showed that FrLRR was primarily distributed in B. belcheri intestinal tissue. We further demonstrated that FrLRR localized to the cell membrane and lysosomes. Functionally, FrLRR mediated and promoted bacteria-binding and phagocytosis, and FrLRR antibody blocking or Grb2 knockdown inhibited FrLRR-mediated phagocytosis. Interestingly, we found that human Slit2-N (hSlit2-N) also mediated direct bacteria-binding and phagocytosis which was inhibited by Slit2-N antibody blocking or Grb2 knockdown. Together, these results indicate FrLRR and hSlit2-N may function as phagocytotic-receptors to promote phagocytosis through Grb2, implying the Slit2-N-type-LRR-containing proteins play a role in bacterial binding and elimination.

Metastasis is the main death reason for triple-negative breast cancer (TNBC). Thus, identifying the driver genes associated with metastasis of TNBC is urgently needed. CRISPR screens have dramatically enhanced genome editing and made it possible to identify genes associated with metastasis. In this study, we identified and explored the crucial role of ras homolog family member V (RhoV) in TNBC metastasis. Here, we performed customized in vivo CRISPR screens targeting metastasis-related genes obtained from transcriptome analysis of TNBC. The regulatory role of RhoV in TNBC was validated using gain- or loss-of-function studies in vitro and in vivo. We further conducted immunoprecipitation and LC-MS/MS to explore the metastasis mechanism of RhoV. In vivo functional screens identified RhoV as a candidate regulator involved in tumor metastasis. RhoV was frequently upregulated in TNBC and correlated with poor survival. Knockdown of RhoV significantly suppressed cell invasion, migration, and metastasis both in vitro and in vivo. In addition, we provided evidence that p-EGFR interacted with RhoV to activate the downstream signal pathway of RhoV, thereby promoting tumor metastasis. We further confirmed that this association was dependent on GRB2 through a specific proline-rich motif in the N-terminus of RhoV. This mechanism of RhoV is unique, as other Rho family proteins lack the proline-rich motif in the N-terminus.

BACKGROUND: Previous data have shown that circ_0033596 is involved in the pathogenesis of atherosclerosis (AS). The study aims to reveal the detailed mechanism of circ_0033596 in AS.
METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to establish an AS cell model. Quantitative real-time polymerase chain reaction and western blot were implemented to detect the expression of circ_0033596, miR-637, growth factor receptor bound protein2 (GRB2), BCL2-associated x protein (Bax) and B-cell lymphoma-2 (Bcl-2). Cell viability, proliferation, apoptosis and tube formation were investigated by cell counting kit-8, EdU assay, flow cytometry and tube formation assay, respectively. The production of interleukin (IL-6) and tumor necrosis factor-α (TNF-α) was evaluated by enzyme-linked immunosorbent assay. Oxidative stress was evaluated by lipid peroxidation malondialdehyde assay kit and superoxide dismutase activity assay kit. Dual-luciferase reporter assay, RNA pull-down assay and RIP assay were performed to identify the associations among circ_0033596, miR-637 and GRB2.
RESULTS: The expression of circ_0033596 and GRB2 was significantly increased, while miR-637 was decreased in the blood of AS patients and ox-LDL-induced HUVECs compared with controls. Ox-LDL treatment inhibited HUVEC viability, proliferation and angiogenic ability and induced cell apoptosis, inflammation and oxidative stress, while these effects were attenuated after circ_0033596 knockdown. Circ_0033596 interacted with miR-637 and regulated ox-LDL-induced HUVEC damage by targeting miR-637. In addition, GRB2, a target gene of miR-637, participated in ox-LDL-induced HUVEC injury by combining with miR-637. Importantly, circ_0033596 activated GRB2 by interacting with miR-637.
CONCLUSIONS: Circ_0033596 depletion protected against ox-LDL-induced HUVEC injury by miR-637/GRB2 pathway, providing a therapeutic target for AS.

Non-small cell lung cancer (NSCLC) is associated with high morbidity and mortality. Dysregulation of lncRNAs leads to NSCLC progression.
This study aims to explore the regulatory mechanism of lncRNA LINC01234 in NSCLC.
LINC01234 expression in NSCLC cells was determined. Cell proliferation was detected using CCK-8, colony formation, and EDU assays after transfection of siRNA LINC01234 into H1299 cells and transfection of pcDNA3.1-LINC01234 into H1975 cells. Subcellular localization of LINC01234 was predicted and the binding relations between LINC01234 and miR-433-3p as well as miR-433-3p and GRB2 were verified. The expression levels of miR-433-3p and GRB2 in NSCLC cells were determined. Joint experiments of miR-433-3p inhibitor + si- LINC01234-1 or oe-GRB2 + si-LINC01234-1 were conducted to verify the role of miR-433-3p and GRB2 in NSCLC cell malignant proliferation.
LINC01234 was abundantly expressed in NSCLC cells. LINC01234 silencing reduced NSCLC cell proliferation while LINC01234 overexpression enhanced cell proliferation. LINC01234 competitively bound to miR-433-3p and miR-433-3p directly targeted GRB2. miR- 433-3p knockdown or GRB2 overexpression counteracted the repressive effect of LINC01234 silencing on NSCLC cell malignant proliferation.
LINC01234 competitively bound to miR-433-3p and promoted GRB2 transcription to augment NSCLC cell malignant proliferation.