Analysis of conformational motions and related key residue interactions responsible for a specific function of proteins with elastic network model

Protein collective motions play a critical role in many biochemical processes. How to predict the functional motions and the related key residue interactions in proteins is important for our understanding in the mechanism of the biochemical processes. Normal mode analysis (NMA) of the elastic network model (ENM) is one of the effective approaches to investigate the structure-encoded motions in proteins. However, the motion modes revealed by the conventional NMA approach do not necessarily correspond to a specific function of protein. In the present work, a new analysis method was proposed to identify the motion modes responsible for a specific function of proteins and then predict the key residue interactions involved in the functional motions by using a perturbation approach. In our method, an internal coordinate that accounts for the specific function was introduced, and the Cartesian coordinate space was transformed into the internal/Cartesian space by using linear approximation, where the introduced internal coordinate serves as one of the axes of the coordinate space. NMA of ENM in this internal/Cartesian space was performed and the function-relevant motion modes were identified according to their contributions to the specific function of proteins. Then the key residue interactions important for the functional motions of the protein were predicted as the interactions whose perturbation largely influences the fluctuation along the internal coordinate. Using our proposed methods, the maltose transporter (MalFGK₂) from E. Coli was studied. The functional motions and the key residue interactions that are related to the channel-gating function of this protein were successfully identified.     

Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.     

A Signaling Pathway Involving the Diguanylate Cyclase CelR and the Response Regulator DivK Controls Cellulose Synthesis in Agrobacterium tumefaciens

The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK, the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR. These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK.     

Acute Myocardial Infarction Risk in Patients with Coronary Artery Disease Doubled after Upper Gastrointestinal Tract Bleeding: A Nationwide Nested Case-Control Study

Prior studies of upper gastrointestinal bleeding (UGIB) and acute myocardial infarction (AMI) are small, and long-term effects of UGIB on AMI have not been delineated. We investigated whether UGIB in patients diagnosed with coronary artery disease (CAD) increased their risk of subsequent AMI. This was a population-based, nested case-control study using Taiwan’s National Health Insurance Research Database. After propensity-score matching for age, gender, comorbidities, CAD date, and follow-up duration, we identified 1,677 new-onset CAD patients with AMI (AMI^[+]) between 2001 and 2006 as the case group and 10,062 new-onset CAD patients without (AMI^[−]) as the control group. Conditional logistic regression was used to examine the association between UGIB and AMI. Compared with UGIB^[−] patients, UGIB^[+] patients had twice the risk for subsequent AMI (adjusted odds ratio [AOR] = 2.08; 95% confidence interval [CI], 1.72–2.50). In the subgroup analysis for gender and age, UGIB^[+] women (AOR = 2.70; 95% CI, 2.03–3.57) and patients < 65 years old (AOR = 2.23; 95% CI, 1.56–3.18) had higher odds of an AMI. UGIB^[+] AMI^[+] patients used nonsignificantly less aspirin than did UGIB^[−] AMI^[+] patients (27.69% vs. 35.61%, respectively). UGIB increased the risk of subsequent AMI in CAD patients, especially in women and patients < 65. This suggests that physicians need to use earlier and more aggressive intervention to detect UGIB and prevent AMI in CAD patients.     

eEF-2 Phosphorylation Down-Regulates P-Glycoprotein Over-Expression in Rat Brain Microvessel Endothelial Cells

ObjectiveWe investigated whether glutamate, NMDA receptors, and eukaryote elongation factor-2 kinase (eEF-2K)/eEF-2 regulate P-glycoprotein expression, and the effects of the eEF-2K inhibitor NH125 on the expression of P-glycoprotein in rat brain microvessel endothelial cells (RBMECs).MethodsCortex was obtained from newborn Wistar rat brains. After surface vessels and meninges were removed, the pellet containing microvessels was resuspended and incubated at 37°C in culture medium. Cell viability was assessed by the MTT assay. RBMECs were identified by immunohistochemistry with anti-vWF. P-glycoprotein, phospho-eEF-2, and eEF-2 expression were determined by western blot analysis. Mdr1a gene expression was analyzed by RT-PCR.ResultsMdr1a mRNA, P-glycoprotein and phospho-eEF-2 expression increased in L-glutamate stimulated RBMECs. P-glycoprotein and phospho-eEF-2 expression were down-regulated after NH125 treatment in L-glutamate stimulated RBMECs.ConclusionseEF-2K/eEF-2 should have played an important role in the regulation of P-glycoprotein expression in RBMECs. eEF-2K inhibitor NH125 could serve as an efficacious anti-multidrug resistant agent.     

Phylogenetic relationships ofPelvetia andPelvetiopsis (Phaeophyceae) based on small subunit ribosomal DNA sequences

Nucleotide sequences of the small-subunit (SSU) ribosomal DNA were determined forPelvetia babingtonii, P. canaliculate, Pelvetiopsis limitata, andAscophyllum nodosum in the family Fucaceae. A total of 1755 positions were aligned for the whole sequence. The positional differences in the primary structure among the taxa ranged from 16 to 30 nucleotide changes in pairwise comparisons. There was a minimum divergence betweenPs. limitata andP. babingtonii while a maximum betweenPs. limitata andP. canaliculata. The SSU rDNA trees showed that the genusPelvetia was not monophyletic and the genusPelvetiopsis was not closely related toPelvetia. Our results suggest that the taxonomic revision of the genusPelvetia as well as the family Fucaceae is needed based on detailed morphological observations.