Expression, purification, and characterization of the histidine kinase CarS from Fusobacterium nucleatum / 生物工程学报

Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.

The Regulation of Lipid Binding and Kinase Activities of EphA2 Kinase Domain by Its Adjacent SAM Domain / 中国生物化学与分子生物学报

The erythropoietin-producing hepatocellular receptor (Eph receptor) family is the largest subfamily in the receptor tyrosine kinase (RTK) families which mediates cell morphology, adhesion, movement, proliferation, survival, and differentiation by its bidirectional signals coupled with Ephrin ligands. EphA2 receptor is an important isoform which is involved in the pathological changes in cataract, breast cancer, etc. Previous studies found that the kinase domain of the EphA2 receptor binds to the plasma membrane, and its kinase activity is regulated by the plasma membrane. However, it is still unclear that the impact of the adjacent SAM domain on the membrane binding and kinase activities of kinase domain. In this study we purified the cytoplasmic kinase-SAM tandem of the EphA2 receptor by co-expression with the phosphatase PTP1B 1-301 fragment. Our results showed that the SAM domain of EphA2 receptor can further enhance the interaction between the kinase domain and liposomes (4 mg/mL) by 6 folds (P<0. 001). And the phosphorylation of kinase-SAM tandem can enhance its lipid (4 mg/mL) binding ability by 2. 5 folds (P < 0. 05). In addition, the lipid binding ability and tyrosine phosphorylation activities of kinase domain are mutual promoted, which creating a positive feedback loop in the two biological processes. In conclusion, our studies indicate that the kinase domain and the adjacent SAM domain can function as an intact unit, whose lipid binding ability and kinase activity are quite different from the individual kinase domain. Therefore, our results provide a biochemical basis for better understanding of the regulation mechanism of other Eph receptors in its kinase domain.

Haploinsufficient tumor suppressor Tip60 negatively regulates oncogenic Aurora B kinase

The Aurora kinases represent a group of serine/threonine kinases which are crucial regulators of mitosis. DysregulatedAurora kinase B (AurkB) expression, stemming from genomic amplification, increased gene transcription or overexpressionof its allosteric activators, is capable of initiating and sustaining malignant phenotypes. Although AurkB level in cells iswell-orchestrated, studies that relate to its stability or activity, independent of mitosis, are lacking. We report that AurkBundergoes acetylation in vitro by lysine acetyltransferases (KATs) belonging to different families, namely by p300 andTip60. The haploinsufficient tumor suppressor Tip60 acetylates two highly conserved lysine residues within the kinasedomain of AurkB which not only impinges the protein stability but also its kinase activity. These results signify a probableoutcome on the increase in ‘‘overall activity’’ of AurkB upon Tip60 downregulation, as observed under cancerous conditions. The present work, therefore, uncovers an important functional interplay between AurkB and Tip60, frailty of whichmay be an initial event in carcinogenesis.

Modulation of nuclear receptor-binding factor 2 on the activity of autophagy initiation complex PI3KC3-C1 / 上海交通大学学报（医学版）

Objective • To study the effects of different phosphorylation levels of nuclear receptor-binding factor 2 (NRBF2) on the activity of class III phosphatidylinositol 3-kinase complex (PI3KC3-C1), and find a type of PI3KC3-C1 with the highest kinase activity as a target for the screening of autophagy-specific inhibitors. Methods • First, in vitro protein purification system was established to obtain quaternary NRBF2-free PI3KC3-C1 and quinary PI3KC3-C1 containing different phosphorylation levels of NRBF2. The integrity of the complex was determined by gel filtration chromatography. The different kinase activity of purified PI3KC3-C1 was detected in vitro, and thus the effect of phosphorylation of NRBF2 on PI3KC3-C1 activity was observed. Results • The purified PI3KC3-C1 had good purity and yield. The five-membered PI3KC3-C1 containing NRBF2 showed higher kinase activity than the quaternary protein complex, and PI3KC3-C1 containing the continuous dephosphorylated NBRF2 had the highest kinase activity. Conclusion • NRBF2 does exist as one of the components of PI3KC3-C1. The presence of NRBF2 promotes the kinase activity of PI3KC3-C1, and PI3KC3-C1 containing continuous dephosphorylation of NRBF2 may serve as a new regulatory target for the screening of autophagy-specific inhibitor.

Antisynthetase syndrome with positive anti-Ro52 antibody: A report of 2 cases and literature review / 吉林大学学报(医学版)

Objective: To summarize the relationship between anti-Ro-52 antibody and anti-synthase syndrome(ASS)by analyzing the diagnosis and treatment of ASS patients with positive anti-Ro-52 antibody, and to clarify its significance. Methods: The clinical materials of 2 ASS patients with positive anti-Ro52 antibody were collected,and the clinical characteristics were analyzed; combined with literature review, the relationship between anti-Ro-52 antibody and ASS was explored.Results: Two young female patients were admitted to hospital because of the obvious symptoms of both hand joints. On physical examination, arthroncus of both hand joints was observed, both hands showed "mechanic hand", muscle strength and muscle tension of limbs were normal. The activity of creatine kinase was increased. Myositis antibody spectrum, lung CT, electromyography and muscle biopsy were performed and the patients were given the related treatments after confirmed diagnosis. The anti-Jo-1 and anti-Ro-52 antibodies were strongly positive in the myositis antibody spectrum of the two patients; the lung CT results showed interstitial pheumonia;the electromyogram results indicated myogenic damage; the results of muscle biopsy was consistent with the changes of idiopathic inflammatory myopathy. Both patients were diagnosed as ASS. After treatment of glucocorticoids and immunosuppressive agents, the symptoms were improved and the creatine kinase activity was decreased. No disease activity and secondary tumor signs were found in the follow-up.Conclusion: As a myositis-associated antibody,anti-Ro-52 antibody can lead to a series of atypical clinical manifestations in the ASS patients and may be associated with the poor prognosis of the ASS patients. Follow-up should be paid attention to.

Kinase activity of novel receptor interacting protein kinase 3 mutants / 上海交通大学学报（医学版）

Objective:To explore the kinase activity of novel receptor interacting protein kinase 3 (RIPK3) mutants. Methods:The four amino acids (Q84WDF87) of RIPK3 were mutated respectively and these mutants were co-transfected with mixed lineage kinase domain like pseudokinase (MLKL) into HEK293T cells. The auto-phosphorylation of these mutants at S232 and phosphorylation of MLKL at S345 were detected by Western blotting. The interaction between RIPK3 and MLKL was tested by co-immunoprecipitation. The oligomerization of MLKL was detected by non-reducing gel. Results:The kinase activities of RIPK3ΔQ84, RIPK3ΔW85 and RIPK3ΔD86 were effectively decreased. Nevertheless, the kinase activities of RIPK3Q84A/RIPK3Q84E, RIPK3W85Y and RIPK3D86A/RIPK3D86Y did not change markedly. The auto-phosphorylation of RIPK3W85A at S232 was decreased without affecting phosphorylation and oligomerization of MLKL. Conclusion:The amino acid site Q84, W85 or D86 plays a critical role in RIPK3 kinase activity. The kinase activity of RIPK3W85A is decreased, but it does not affect MLKL.

A mechanism research of novel inhibitor of PAK1 inducing colorectal cancer DLD-1 apoptosis / 中国药科大学学报

@#PAK1 plays an important role in the development of tumors. It is of great significance to screen and develop new PAK1 inhibitors as targeted drugs for cancer treatment. The traditional PAK1 inhibitor screening method has the problems of high cost and low efficiency. Computer virtual screening can reduce the cost of finding active lead compounds and improve the screening efficiency. In this study, a kind of PAK1 candidate compound was screened by computer assisted virtual screening combined with Z′lyteTM high flux kinase screen. In vitro enzyme activity screening showed that compound 18(K788)had good PAK1 inhibitory activity(inhibition rate was 42. 7%). Furtherly by MTT detection, it was found that K788 had significant PAK1 positive tumor killing activity, which was even better than the positive drug IPA-3. Flow cytometry and Western Blot showed that K788 could activate caspase apoptosis pathway and induce apoptosis of colon cancer cell DLD-1 by inhibiting PAK1 expression and activation. K788 has great potential for clinical development and application, and can be used as a PAK1 target for further research.

Effects of substrate stiffness on confluent growth of hepatic and hepatoma carcinoma cells / 医用生物力学

Objective To investigate the cause of differences in confluent growth between hepatic(L02) and hepatoma carcinoma(HCCLM3) cells by comparing responses of the two cells to different substrate stiffness (0.5, 4 kPa and glass). MethodsThe real-time photomicrography, immunofluorescence staining, flow cytometry, and Western Blotting techniques were respectively employed to observe the morphological characteristics, the cytoskeleton conformation and the distribution of E-cad of confluent L02 and HCCLM3 cells on different substrates, and test the changes in expression of E-cad, Integrinβ1 and p-Src. Results (1) Confluent L02 cells displayed a round or cubic shape, while HCCLM3 cells showed a polygon shape. The morphology of HCCLM3 cells were spread and polarized more obviously than that of L02 cells. With the increase of substrate stiffness, the variation of L02 cells with time was smaller than that of HCCLM3 cells. (2) The cytoskeleton of confluent L02 cells showed a ring-like conformation under the cortex, and E-cad was located at the cell-cell contact sites. However, the ring-like cytoskeleton of HCCLM3 cells was incomplete and distributed radially along the basement, while E-cad was dispersed in cytoplasm. (3) As the substrate stiffness increased, expression of E-cadherin in both L02 and HCCLM3 cells was significantly decreased (P<0.01), while the level of p-Src and integrinβ1 was increased significantly, with greater changes in HCCLM3 cells than in L02 cells. Conclusions The assembling of cortical ring-like cytoskeleton was positively correlated with the location of E-cad at the cell-cell contact sites. The substrate stiffness showed a more obvious impact on the balanced regulation between cadherin and integrin mediated adhesion system of hepatocarcinoma cells than that of hepatic cells.

Reduced Serum Creatine Kinase Activity in Patients with Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: Our objective was (1) to determine if serum creatine kinase (CK) activity is reduced in rheumatoid arthritis (RA) compared with that of noninflammatory rheumatic diseases, (2) to examine the recently described association of low CK activity and disease variables in our RA population, and (3) to examine the influence of steroid on serum CK activity in patients with RA. METHODS: Cross sectional and longitudinal retrospective analyses of clinical and biochemical data of consecutive patients with RA and noninflammatory arthropathies. In all subjects we evaulated age, sex, weight, and, only for patients with RA, history of use of corticosteroids and Ritchie index. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), hemoglobin, and platelet count were simultaneously determined as variables of disease activity. CK activity was determined by automated biochemical analyzer (Hitachi 747, Japan). RESULTS: Serum CK activity was significantly reduced in RA (mean+SD: 45.7 +24.2 IU/L) compared to controls (81.3+33.9 IU/L) (p < 0.001). Ritchie index, CRP, and platelet count correlated inversely with CK values (correlation coefficient: 0.31, p < 0.01; 0. 45, p < 0.001; 0.42, p < 0.001, respectively). Patients taking steroids had lower CK activity than those without steroid, but not statistically significant.

Effects of repeated exercise on muscle soreness, creatine kinase activity and white blood cell count / 体力科学

A study was conducted to investigate changes in muscle soreness, serum creatine kinase (CK) activity and white blood cell (WBC) count following exercise bouts spaced three weeks apart.<BR>The subjects were six male students (aged 23-25 yr), who had not participated in any training program for over 18 months. They performed muscular exercise of the nondominant arm using elbow flexors. Twenty percent of maximum voluntary contraction was used as the exercise intensity. After three weeks, the subjects repeated the same exercise bout. Perceived muscle soreness, CK activity and WBC count were assessed before, immediately after, 6h after and over 9 days after each exercise bout.<BR>After the first exercise bout (1 st Ex), the subjects experienced muscle sorenss for 3-7 days. Also, a large increase of CK was found in five subjects (266-763%) . When the peak CK efflux was observed (day 3-4 after exercise), soreness had almost disappeared. WBC count was increased immediately and 6 h after exercise, then returned to the resting level. However, a significant increase (p<0.05) in WBC count was observed again on day 7 after exercise when CK had returned to the resting level. After the second exercise (2 nd Ex), a significant decrease of muscle soreness and the CK response was found in comparison with the 1 st Ex (p<0.41) . One interesting feature was that the CK efflux of subjects who had shown a large increase of CK after the 1 st Ex was not increased after the 2 nd Ex.<BR>The initial exercise bout may have induced some damage to the muscle fibers or mem. bran. This damage would induce a process of repair in the damaged tissue, which in turn would adapt the muscle to the next stimulus. However, the subjects who showed a slight increase of CK after the 1 st Ex did not show this adaptation. Therefore an adaptive threshold for fiber or membrane damage may exist.