Cell Division Control Protein Research Articles

Pathways and molecules for overcoming immunotolerance in metastatic gastrointestinal tumors.

Worldwide, gastrointestinal (GI) cancer is recognized as one of the leading malignancies diagnosed in both genders, with mortality largely attributed to metastatic dissemination. It has been identified that in GI cancer, a variety of signaling pathways and key molecules are modified, leading to the emergence of an immunotolerance phenotype. Such modifications are pivotal in the malignancy's evasion of immune detection. Thus, a thorough analysis of the pathways and molecules contributing to GI cancer's immunotolerance is vital for advancing our comprehension and propelling the creation of efficacious pharmacological treatments. In response to this necessity, our review illuminates a selection of groundbreaking cellular signaling pathways associated with immunotolerance in GI cancer, including the Phosphoinositide 3-kinases/Akt, Janus kinase/Signal Transducer and Activator of Transcription 3, Nuclear Factor kappa-light-chain-enhancer of activated B cells, Transforming Growth Factor-beta/Smad, Notch, Programmed Death-1/Programmed Death-Ligand 1, and Wingless and INT-1/beta-catenin-Interleukin 10. Additionally, we examine an array of pertinent molecules like Indoleamine-pyrrole 2,3-dioxygenase, Human Leukocyte Antigen G/E, Glycoprotein A Repetitions Predominant, Clever-1, Interferon regulatory factor 8/Osteopontin, T-cell immunoglobulin and mucin-domain containing-3, Carcinoembryonic antigen-related cell adhesion molecule 1, Cell division control protein 42 homolog, and caspases-1 and -12.

Kisspeptin/KISS1R Signaling Modulates Human Airway Smooth Muscle Cell Migration.

Airway remodeling is a cardinal feature of asthma, associated with increased airway smooth muscle cell (ASM) mass and up-regulation of extracellular matrix deposition. Exaggerated ASM cell migration contributes to excessive ASM mass. Previously, we demonstrated the alleviating role of kisspeptin (Kp) receptor (KISS1R) activation by Kp-10 in mitogen (PDGF)-induced human ASM cell proliferation in vitro and airway remodeling in vivo in a mouse model of asthma. Here, we examined the mechanisms by which KISS1R activation regulates mitogen-induced ASM cell migration. KISS1R activation using Kp-10 significantly inhibited PDGF-induced ASM cell migration, further confirmed using KISS1R shRNA. Furthermore, KISS1R activation modulated F/G actin dynamics and the expression of pro-migration proteins like cell division control protein 42 (CDC42) and cofilin. Mechanistically, we observed reduced ASM RhoA-GTPAse with KISS1R activation. The anti-migratory effect of KISS1R was abolished by protein kinase A (PKA)-inhibitory peptide. Conversely, KISS1R activation significantly increased cAMP and phosphorylation of cAMP-response element binding protein (CREB) in PDGF-exposed ASM cells. Overall, these results highlight the alleviating properties of Kp-10 in the context of airway remodeling.

Gut commensal metabolite rhamnose promotes macrophages phagocytosis by activating SLC12A4 and protects against sepsis in mice

Sepsis progression is significantly associated with the disruption of gut eubiosis. However, the modulatory mechanisms of gut microbiota operating during sepsis are still unclear. Herein, we investigated how gut commensals impact sepsis development in a pre-clinical model. Cecal ligation and puncture (CLP) surgery was used to establish polymicrobial sepsis in mice. Mice depleted of gut microbiota by an antibiotic cocktail (ABX) exhibited a significantly higher level of mortality than controls. As determined by metabolomics analysis, ABX treatment has depleted many metabolites, and subsequent supplementation with l-rhamnose (rhamnose, Rha), a bacterial carbohydrate metabolite, exerted profound immunomodulatory properties with a significant enhancement in macrophage phagocytosis, which in turn improved organ damage and mortality. Mechanistically, rhamnose binds directly to and activates the solute carrier family 12 (potassium-chloride symporter), member 4 (SLC12A4) in macrophages and promotes phagocytosis by activating the small G-proteins, Ras-related C3 botulinum toxin substrate1 (Rac1) and cell division control protein 42 homolog (Cdc42). Interestingly, rhamnose has enhanced the phagocytosis capacity of macrophages from sepsis patients. In conclusion, by identifying SLC12A4 as the host interacting protein, we disclosed that the gut commensal metabolite rhamnose is a functional molecular that could promote the phagocytosis capacity of macrophages and protect the host against sepsis.

Connecting the ends: signaling via receptor tyrosine kinases and cytoskeletal degradation in neurodegeneration

Receptor tyrosine kinases (RTKs) are known to perform versatile roles in disease landscapes, which determine the fate of the cell. Although much has been discussed from the perspective of proliferation, this review focuses on the impact of RTK-mediated signaling and its role in cytoskeletal degradation, the penultimate stage of cellular degeneration. In the case of degenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), age-related macular degeneration (AMD), and type 2 diabetes mellitus (T2DM), RTK signaling has been reported to be perturbed in several studies. The implications of downstream signaling via these receptors through canonical and noncanonical pathways alter the status of actin filaments that provide structural integrity to cells. Degenerative signaling leads to the altered status of rat sarcoma (Ras), Ras homologous (Rho), Ras-related C3 botulinum toxin substrate (Rac), and cell division control protein 42 (Cdc42), the best-characterized components of the cytoskeleton remodeling machinery. RTKs, along with their diverse adaptor partners and other membrane receptors, affect the functionality of Rho family guanosine triphosphate hydrolases (GTPases), which are discussed in this review. To conclude, this review focuses on therapeutic strategies targeting RTKs and Rho GTPase-mediated pathways that can be more effective due to their combined multifactorial impact on neurodegenerative cascades.

Retracted] microRNA‑195 inhibits cell proliferation in bladder cancer via inhibition of cell division control protein 42 homolog/signal transducer and activator of transcription‑3 signaling.

[This retracts the article DOI: 10.3892/etm.2015.2633.].

Modified Danzhi Xiaoyao Powder (MDXP) improves the corneal damage in dry eye disease (DED) mice through phagocytosis

Ethnopharmacological relevanceModified Danzhi Xiaoyao Powder (MDXP) is a traditional Chinese medicine formula remedy for treating Dry Eye Disease (DED). It showed the function of dispersing stagnated liver Qi for relieving Qi stagnation and clearing heat, which can be effective in treating conditions such as Dry Eye Disease (DED) and irregular menstruation due to liver depression and fire transformation. Aim of the studyThis study investigated the mechanism of the effect of MDXP in mice with DED. Materials and methodsA DED model was induced in mice using chronic painful stimulation (tail clamping) in combination with Benzalkonium Chloride Solution drops administered in a dry box for 28 days. After modeling, the MDXP groups were given Chinese medicine with different dosages by gavage for 14 days. The following parameters were recorded in each group: body mass, anal temperature, tear secretion, tear film rupture time, and corneal fluorescein staining. Behavioral changes were evaluated by elevating cross-maze and open-field experiments. The levels of inflammatory factors serum tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), fcγR-mediated phagocytosis pathway cell division control protein 42 homolog (CDC42), actin-related protein 2/3 complex subunit 2 (ARPC2), and actin-related protein 3 (ACTR3) were measured by using Enzyme-linked immunoassay (ELISA), immunohistochemical staining, and real-time fluorescent qualitative polymerase chain reaction (RT-qPCR). ResultsMDXP increased body mass and lowered body temperature, prolonged tear film break-up time, promoted tear secretion, repaired corneal damage, decreased horizontal and vertical scores, elevated percentage of open arm times and boom opening time percentage, and reduced the expression levels of inflammatory factors of TNF-α, IL-1β and pathway-related proteins CDC42, ARPC2, and ACTR3 in mice. MDXP also reduced the expression levels of inflammatory factors of TNF-α and IL-1β in human corneal endothelial cells (HCECs), mouse mononuclear macrophage cells (RAW264.7), and human myeloid leukemia mononuclear cells (THP-1). ConclusionsMDXP can relieve tension and anxiety, inhibit apoptosis, reduce phagocytosis, reduce the expression of pro-inflammatory factors, repair corneal damage, and improve the symptoms in DED mice. The mechanism of action may be through the fcγR-mediated phagocytosis pathway.

LINC00869 Promotes Hepatocellular Carcinoma Metastasis via Protrusion Formation.

LncRNA LINC00869 regulates the activity of CDC42-WASP pathway and positively affects protrusion formation in HCC cells, which expands the current understanding of lncRNA functions as well as gives a better understanding of carcinogenesis.

Monotropein Induced Apoptosis and Suppressed Cell Cycle Progression in Colorectal Cancer Cells.

To determine whether monotropein has an anticancer effect and explore its potential mechanisms against colorectal cancer (CRC) through network pharmacology and molecular docking combined with experimental verification. Network pharmacology and molecular docking were used to predict potential targets of monotropein against CRC. Cell counting kit assay, plate monoclonal assay and microscopic observation were used to investigate the antiproliferative effects of monotropein on CRC cells HCT116, HT29 and LoVo. Flow cytometry and scratch assay were used to analyze apoptosis and cell cycle, as well as cell migration, respectively in HCT116, HT29, and LoVo cells. Western blotting was used to detect the expression of proteins related to apoptosis, cell cycle, and cell migration, and the expression of proteins key to the Akt pathway. The Gene Ontology and Reactome enrichment analyses indicated that the anticancer potential of monotropein against CRC might be involved in multiple cancer-related signaling pathways. Among these pathways, RAC-beta serine/threonine-protein kinase (Akt1, Akt2), cyclin-dependent kinase 6 (CDK6), matrix metalloproteinase-9 (MMP9), epidermal growth factor receptor (EGFR), cell division control protein 42 homolog (CDC42) were shown as the potential anticancer targets of monotropein against CRC. Molecular docking suggested that monotropein may interact with the 6 targets (Akt1, Akt2, CDK6, MMP9, EGFR, CDC42). Subsequently, cell activity of HCT116, HT29 and LoVo cell lines were significantly suppressed by monotropein (P<0.05). Furthermore, our research revealed that monotropein induced cell apoptosis by inhibiting Bcl-2 and increasing Bax, induced G1-S cycle arrest in colorectal cancer by decreasing the expressions of CyclinD1, CDK4 and CDK6, inhibited cell migration by suppressing the expressions of CDC42 and MMP9 (P<0.05), and might play an anticancer role through Akt signaling pathway. Monotropein exerts its antitumor effects primarily by arresting the cell cycle, causing cell apoptosis, and inhibiting cell migration. This indicates a high potential for developing novel medication for treating CRC.

C-terminal variants in CDC42 drive type I interferon-dependent autoinflammation in NOCARH syndrome reversible by ruxolitinib

C-terminal variants in CDC42 encoding cell division control protein 42 homolog underlie neonatal-onset cytopenia, autoinflammation, rash, and hemophagocytic lymphohistiocytosis (NOCARH). Pyrin inflammasome hyperactivation has been shown to contribute to disease pathophysiology. However, mortality of NOCARH patients remains high despite inflammasome-focused treatments. Here, we demonstrate in four NOCARH patients from three families that cell-intrinsic activation of type I interferon (IFN) is a previously unrecognized driver of autoinflammation in NOCARH. Our data show that aberrant innate immune activation is caused by sensing of cytosolic nucleic acids released from mitochondria, which exhibit disturbances in integrity and dynamics due to CDC42 dysfunction. In one of our patients, treatment with the Janus kinase inhibitor ruxolitinib led to complete remission, indicating that inhibition of type I IFN signaling may have an important role in the management of autoinflammation in patients with NOCARH.

β-hydroxybutyrate impairs the directionality of migrating neutrophils through inhibiting the autophagy-dependent degradation of Cdc42 and Rac1 in ketotic cows

Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly β-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophil need further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB <0.6 mM) or clinical ketosis (n = 20, BHB >3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

Serum CDC42 is increased during tumor necrosis factor inhibitor treatment, and its elevation correlates with satisfactory treatment response in rheumatoid arthritis patients.

Our previous study discovered that cell division control protein 42 (CDC42) correlated with decreased disease activity and risk of developing rheumatoid arthritis (RA), along with repressed T helper type 17 cell differentiation. This study aimed to further estimate the longitudinal change of serum CDC42 and its association with treatment outcomes to tumor necrosis factor inhibitor (TNFi) in RA. CDC42 was detected in serum by ELISA at week (W)0, W6, W12, and W24 in 88 RA patients undergoing TNFi treatment, and after enrollment in 20 disease controls (DCs) and 20 healthy controls (HCs). CDC42 was lower in RA patients compared with DCs and HCs (both p < .001); meanwhile, it negatively related to C-reactive protein (p = .011) and DAS28 score (p = .006). Regarding TNFi type, 40.9%, 33.0%, 17.0%, and 9.1% of patients received adalimumab, etanercept, golimumab, and infliximab, respectively. Notably, CDC42 was increased from W0 to W24 in RA patients receiving TNFi treatment (p < .001), also in patients receiving adalimumab (p < .001), etanercept (p < .001), golimumab (p < .001), and infliximab (p = .001). Furthermore, CDC42 at W24 was higher in patients with a clinical response to TNFi treatment compared with those without (p = .023); CDC42 at W12 (p = .027) and W24 (p = .002) was elevated in patients with clinical low disease activity in response to TNFi treatment versus those without; whereas CDC42 at W12 (p = .074) and W24 (p = .068) only showed an increasing trend in patients with clinical remission with TNFi treatment, but did not achieve statistical significance. Circulating CDC42 is elevated during TNFi administration; its increase reflects good 24-week TNFi treatment responses in RA patients.

ICP4-Associated Activation of Rap1b Facilitates Herpes Simplex Virus Type I (HSV-1) Infection in Human Corneal Epithelial Cells.

The strong contribution of RAS-related protein 1b (Rap1b) to cytoskeleton remodeling determines intracellular and extracellular physiological activities, including the successful infection of viruses in permissive cells, but its role in the HSV-1 life cycle is still unclear. Here, we demonstrated that the HSV-1 immediate early (IE) gene ICP4 inhibits protein kinase A (PKA) phosphorylation to induce Rap1b-activation-mediated viral infection. Rap1b activation and membrane enrichment begin at the early stage of HSV-1 infection and remain active during the proliferation period of the virus. Treating the cells with Rap1b small interfering RNA (siRNA) showed a dose-dependent decrease in viral infection levels, but no dose-dependent increase was observed after Rap1b overexpression. Further investigation indicated that the suppression of Rap1b activation derives from phosphorylated PKA and Rap1b mutants with partial or complete prenylation instead of phosphorylation, which promoted viral infection in a dose-dependent manner. Furthermore, the PKA agonist Forskolin disturbed Rap1b activation in a dose-dependent manner, accompanied by a decreasing trend in viral infection. Moreover, the HSV-1 IE gene ICP4 induced PKA dephosphorylation, leading to continuous Rap1b activation, followed by cytoskeleton rearrangement induced by cell division control protein 42 (CDC42) and Ras-related C3 botulinum toxin substrate 1 (RAC1). These further stimulated membrane-triggered physiological processes favoring virus infection. Altogether, we show the significance of Rap1b during HSV-1 infection and uncover the viral infection mechanism determined by the posttranslational regulation of the viral ICP4 gene and Rap1b host protein.

Methylated dialkylphosphate metabolites of the organophosphate pesticide malathion modify actin cytoskeleton arrangement and cell migration via activation of Rho GTPases Rac1 and Cdc42

The non-cholinergic molecular targets of organophosphate (OP) compounds have recently been investigated to explain their role in the generation of non-neurological diseases, such as immunotoxicity and cancer. Here, we evaluated the effects of malathion and its dialkylphosphate (DAP) metabolites on the cytoskeleton components and organization of RAW264.7 murine macrophages as non-cholinergic targets of OP and DAPs toxicity. All OP compounds affected actin and tubulin polymerization. Malathion, dimethyldithiophosphate (DMDTP) dimethylthiophosphate (DMTP), and dimethylphosphate (DMP) induced elongated morphologies and the formation of pseudopods rich in microtubule structures, and increased filopodia formation and general actin disorganization in RAW264.7 cells and slightly reduced stress fibers in the human fibroblasts GM03440, without significantly disrupting the tubulin or vimentin cytoskeleton. Exposure to DMTP and DMP increased cell migration in the wound healing assay but did not affect phagocytosis, indicating a very specific modification in the organization of the cytoskeleton. The induction of actin cytoskeleton rearrangement and cell migration suggested the activation of cytoskeletal regulators such as small GTPases. We found that DMP slightly reduced Ras homolog family member A activity but increased the activities of Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) from 5 min to 2 h of exposure. Chemical inhibition of Rac1 with NSC23766 reduced cell polarization and treatment with DMP enhanced cell migration, but Cdc42 inhibition by ML-141 completely inhibited the effects of DMP. These results suggest that methylated OP compounds, especially DMP, can modify macrophage cytoskeleton function and configuration via activation of Cdc42, which may represent a potential non-cholinergic molecular target for OP compounds.

Distribution of CNF1among Escherichia coli isolates from urinary tract infection and bladder cancer in southern of Iraq

One of the most important virulence factors produced by uropathogenicEscherichia coli (UPEC) is cytotoxic necrotizing factor 1 (CNF1). It plays a vital role in regulation of Ras homolog family member A (Rho), Ras-related C3 botulinum toxin substrate (Rac), and Cell division control protein 42 homolog (Cdc42)guanosine triphosphatase (GTPases) proteins, which involved in the organization of the actin cytoskeleton in eukaryotic cells. Also, CNF1may play a role in cell proliferation, surviving and gene transcription. Therefore, detection of the gene encoding CNF1 in UPEC would be imperative in characterization of these organisms and establishing a proper management regime to prevent the poor prognosis of the associated diseases. The aim of the present study was to investigatethe prevalence of the gene encoding cnf1 in UPEC strainsthat have been isolated from urinary tract infection and bladder cancer patients.Out of three hundred and fifty midstream urine samples, 136E. colistrains were isolated (98 urinary tract infection, 18 bladder cancer, and 20 healthy people). Detection and determination of the gene encoding CNF1 based on PCR technique and sequencing of PCR amplified target DNA. The present results showed that thirty five percent (35.34%) of the tested isolates have cnf1gene. All cnf1+strains were isolated from urinary tract infection patients. This finding should bring attention to predict the prognosis of the infections caused by these organisms and follow up patients to prevent any further complications. &#x0D;

Abstract 877: Protein-protein interactions in cancer: An in-silico analysis of Cdc6 interactions

Abstract Aberrant activity of cell cycle proteins is a hallmark of cancer and therefore, cell cycle regulators are considered attractive targets in cancer therapy. One such regulator, Cell division cycle 6 (Cdc6), is a key conserved cell cycle protein responsible for the initiation of eukaryotic DNA replication. Cdc6 overexpression has been observed as an early event in malignancies. Additionally, Cdc6 has also been implicated in contributing to the aggressiveness of cervical and bladder cancers. In budding yeast, Cdc6 collaborates with proteins such as Cyclin B2 (Clb2) and Cell division control protein 4 (Cdc4) to ensure the fidelity of cell division by preventing re-replication. The perfectly orchestrated sequence of events requires Cdc6 to interact with other cell cycle proteins during the correct stage of the cell cycle. During S-phase, Cyclin-dependent kinase 1 (Cdk1) regulates Cdc6 levels and Cdc6 is targeted for degradation by polyubiquination via Cdc4 (SCF-complex). In mitosis, phosphorylation by Cdk1, induces the binding of Cdc6 to Clb2-Cdk1. Clb2 binds to Cdc6’s 44PEKLQF49 motif at the Cdc6 N-terminus thereby preventing Cdc6 from licensing origins, preventing DNA re-replication, and ensuring genomic integrity. Although, it has been well studied how each Cdc6 phosphorylation site regulates protein binding, in the absence of full-length structures for many of these proteins, the interaction modes and molecular details for Cdc6’s interaction with its protein partners remain unknown. In this study, we have characterized and analyzed Cdc6’s interactions with some of its key interactors. We present a robust computational approach to understand the molecular mechanisms of Cdc6 interactions with Cdc4 and Clb2, which includes homology/ab initio modeling of the full-length structures including their intrinsically disordered regions (IDRs), and predicted interaction scenarios. Our results show that the Cdc6-Cdc4 interaction displays a dynamic shift between S-phase and mitosis. In S-phase, Cdc4 binds to the N’ phosphodegrons on Cdc6, but in mitosis, Cdc4 binding shifts to the C’ phosphodegrons. In addition, we show Clb2’s K270, within its hydrophobic patch, binds E45 in Cdc6’s 44PEKLQF49 motif. We also provide a comparison of Clb2’s interaction with Cdc6 with that of Bud3 to assess the conservation of the binding mechanism. We show that the molecular mechanism is conserved with similar residues and motifs being involved in the interaction. By exploring the molecular details underlying these protein-protein interactions, we can gain insightful information and identify interface residues that are responsible for specific protein-protein interactions. Such molecular details are critical to pave the way to the discovery of druggable targets and ultimately lead to the development of new cancer therapeutics. Citation Format: Andriele Silva Eichner, Jasmin Philip, Amy Ikui, Shaneen Singh. Protein-protein interactions in cancer: An in-silico analysis of Cdc6 interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 877.

Allosteric inactivation of an engineered optogenetic GTPase

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/"lit" or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light-or alternatively, mutation in LOV2 to mimic light absorption-allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus "lit" states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42's downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.

Long Non-Coding RNA BNIP3 Inhibited the Proliferation of Bovine Intramuscular Preadipocytes via Cell Cycle

The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5' RACE and 3' RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5'RACE, and 464 bp of 3'RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2'- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality.

Lamprey prohibitin 2 inhibits non-small cell lung carcinoma cell proliferation by down-regulating the expression and phosphorylation levels of cell cycle-associated proteins

Prohibitin 2 (PHB2) is an evolutionarily conserved and functionally diverse protein that plays an important role in multiple cellular functions, including cell proliferation, cell migration, and apoptosis, and is also known to participate in the process of tumorigenesis and development. In this study, the lamprey PHB2 (Lm-PHB2) gene was over-expressed in KRAS (kirsten rat sarcoma viral oncogene homolog)-mutated non-small cell lung carcinoma (NSCLC) cells to investigate its effect on cell proliferation. The effects of Lm-PHB2 protein on the proliferation of NSCLC cells were determined by treating cells with the purified recombinant Lm-PHB2 protein (rLm-PHB2) followed by cell counting kit (CCK) assays and flow cytometry. Analysis showed that rLm-PHB2 blocked cells in the G2 phase and inhibited the cell proliferation of A549, Calu-1, and NCI-H226 to various degrees. The effect on Calu-1 cells was the most obvious and was concentration- and time-dependent. Similarly, cells transfected with the pEGFP-N1-Lm-PHB2 plasmid also resulted in the suppression of proliferation in A549 cells and Calu-1 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that Lm-PHB2 inhibited cell proliferation by repressing the transcription of PLK1 (polo-like kinase 1), Wee1 (wee1 kinase), CCNB1 (cyclin B1), and CDC25C (cell division control protein 25C). According to western blot analysis, Lm-PHB2 not only down-regulated the expression of PLK1, Wee1, CCNB1, and CDC25C but also reduced the phosphorylation levels of CCNB1 and CDC25C, thus blocking Calu-1 cells in G2/M phase. Our findings demonstrate a function of lamprey PHB2 that may inhibit the proliferation of some NSCLC cells by down-regulating the expression and phosphorylation of cell cycle-associated proteins.

Cell Division Control Protein 42 Facilitates Diabetic Retinopathy Progression by Activating the MEK/ERK Pathway.

Cell division control protein 42 (CDC42) modulates insulin secretion and angiogenesis to participate in the pathology of diabetic complications and retinal vascular-associated diseases. This study intended to explore the role of CDC42 in the progression of diabetic retinopathy, and the underlying mechanism. Human retinal microvascular endothelial cells (hRMECs) were cultured in 5.5 mM glucose (normal glucose) or 25 mM glucose (high glucose; HG) medium, respectively. CDC42 overexpression plasmid and small interference RNA (oe-CDC42 and si-CDC42) or corresponding negative controls (oe-NC and si-NC) were transfected into hRMECs under HG. Then, platelet-activating factor C-16 (C16-PAF) (MEK/ERK pathway activator) was added to si-CDC42 or si-NC transfected hRMECs under HG. Our study showed that HG increased CDC42 mRNA and protein, cell viability, invasive cell count, branch points, and tube length but reduced cell apoptosis in hRMECs. CDC42 upregulation enhanced cell viability, invasive cell count, branch points, tube length, p-MEK, and p-ERK, but attenuated cell apoptosis. Downregulation of CDC42 exhibited opposite trends. In addition, C16-PAF also increased cell viability, invasive cell count, branch points, and tube length, p-MEK, and p-ERK, but retarded cell apoptosis. Notably, C16-PAF diminished the effect of CDC42 downregulation on the above-mentioned functions in hRMECs under HG. Conclusively, CDC42 promotes HG-induced hRMEC viability and invasion, as well as angiogenesis, but inhibits apoptosis by activating the MEK/ERK pathway, which may be responsible for the progression of diabetic retinopathy.

Elevated barometric pressure suppresses cell proliferation by delaying the G2/M phase and weakening integrin-mediated cell adhesion and actin assembly

Human cells are continuously exposed to various stress factors in their physiological environment. Evidence suggests that certain mechanical stress can affect cell cycle progression and cell proliferation. However, the signaling pathways involved in this process are not well understood. To investigate this, we developed a pressure chamber capable of producing an elevated barometric pressure (EBP) environment of 2?atmospheric absolute pressure (ATA). We then studied the effect of EBP on cell proliferation and its underlying mechanism. Our results show that EBP inhibited cell proliferation by delaying the G2/M phase. Specifically, EBP reduced the expression levels of cell adhesion-related genes and downregulated integrin subunit genes, resulting in weaker interaction between cells and extracellular matrix proteins. In addition, Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 homolog (Cdc42) activity was suppressed, and actin assembly was decreased. These findings suggest that the EBP-mediated G2/M phase delay is due to attenuated cell adhesion and actin cytoskeleton assembly, leading to the inhibition of cell proliferation. Our results provide a crucial molecular mechanism for how certain pressure (changes) can negatively regulate cell proliferation. These findings could potentially be used in the future to develop a pressure therapy to inhibit cell proliferation in cancer patients.