Protein unfolding transitions in an intrinsically unstable annexin domain: molecular dynamics simulation and comparison with nuclear magnetic resonance data

Unfolding transitions of an intrinsically unstable annexin domain and the unfolded state structure have been examined using multiple approximately 10-ns molecular dynamics simulations. Three main basins are observed in the configurational space: native-like state, compact partially unfolded or intermediate compact state, and the unfolded state. In the native-like state fluctuations are observed that are nonproductive for unfolding. During these fluctuations, after an initial loss of approximately 20% of the core residue native contacts, the core of the protein transiently completely refolds to the native state. The transition from the native-like basin to the partially unfolded compact state involves approximately 75% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate state adopts for part of the time in one of the trajectories a novel highly compact salt-bridge stabilized structure that can be identified as a conformational trap. The intermediate-to-unfolded state transition is characterized by a large increase in the radius of gyration. After an initial relaxation the unfolded state recovers a native-like topology of the domain. The simulated unfolded state ensemble reproduces in detail experimental nuclear magnetic resonance data and leads to a convincing complete picture of the unfolded domain.     

Conserved sequence in the aggrecan interglobular domain modulates cleavage by ADAMTS-4 and ADAMTS-5

Background

Cleavage of aggrecan by ADAMTS proteinases at specific sites within highly conserved regions may be important to normal physiological enzyme functions, as well as pathological degradation.

Methods

To examine ADAMTS selectivity, we assayed ADAMTS-4 and -5 cleavage of recombinant bovine aggrecan mutated at amino acids N-terminal or C-terminal to the interglobular domain cleavage site.

Results

Mutations of conserved amino acids from P18 to P12 to increase hydrophilicity resulted in ADAMTS-4 cleavage inhibition. Mutation of Thr, but not Asn within the conserved N-glycosylation motif Asn-Ile-Thr from P6 to P4 enhanced cleavage. Mutation of conserved Thr residues from P22 to P17 to increase hydrophobicity enhanced ADAMTS-4 cleavage. A P4′ Ser377Gln mutant inhibited cleavage by ADAMTS-4 and -5, while a neutral Ser377Ala mutant and species mimicking mutants Ser377Thr, Ser377Asn, and Arg375Leu were cleaved normally by ADAMTS-4. The Ser377Thr mutant, however, was resistant to cleavage by ADAMTS-5.

Conclusion

We have identified multiple conserved amino acids within regions N- and C-terminal to the site of scission that may influence enzyme–substrate recognition, and may interact with exosites on ADAMTS-4 and ADAMTS-5.

General significance

Inhibition of the binding of ADAMTS-4 and ADAMTS-5 exosites to aggrecan should be explored as a therapeutic intervention for osteoarthritis.     

Biochemical differences in Cannabis sativa L. depending on sexual phenotype

Hemp (Cannabis sativa L.) is a species considered as having one of the most complicated mechanisms of sex determination. Peroxidase and esterase isoenzymes in leaves of the two sexual phenotypes of hemp were studied. Significant differences in isoperoxidase and isoesterase patterns were found between male and female plants, both in the number and stain intensity of bands. For both esterase and peroxidase, the isoenzymatic spectrum is richer for staminate plants. Also, some differences are obvious between the two sexes concerning catalase and peroxidase activities, as well as the level of soluble protein. The quantitative analysis of flavones, polyholozides and polyphenols emphasized differences depending not only on sex, but also on tested organ.     

Lichen Secondary Metabolite,Physciosporin, Inhibits Lung Cancer Cell Motility

Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3’-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action.     

Strategy Choice Mediates the Link between Auditory Processing and Spelling

Relations among linguistic auditory processing, nonlinguistic auditory processing, spelling ability, and spelling strategy choice were examined. Sixty-three undergraduate students completed measures of auditory processing (one involving distinguishing similar tones, one involving distinguishing similar phonemes, and one involving selecting appropriate spellings for individual phonemes). Participants also completed a modified version of a standardized spelling test, and a secondary spelling test with retrospective strategy reports. Once testing was completed, participants were divided into phonological versus nonphonological spellers on the basis of the number of words they spelled using phonological strategies only. Results indicated a) moderate to strong positive correlations among the different auditory processing tasks in terms of reaction time, but not accuracy levels, and b) weak to moderate positive correlations between measures of linguistic auditory processing (phoneme distinction and phoneme spelling choice in the presence of foils) and spelling ability for phonological spellers, but not for nonphonological spellers. These results suggest a possible explanation for past contradictory research on auditory processing and spelling, which has been divided in terms of whether or not disabled spellers seemed to have poorer auditory processing than did typically developing spellers, and suggest implications for teaching spelling to children with good versus poor auditory processing abilities.     

Mammalian expression of full-length bovine aggrecan and link protein: formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13

Aggrecan, a large chondroitin sulfate (CS) and keratan sulfate (KS) proteoglycan, has not previously been expressed as a full-length recombinant molecule. To facilitate structure/function analysis, we have characterized recombinant bovine aggrecan (rbAgg) and link protein expressed in COS-7 cells. We demonstrate that C-terminally truncated rbAgg was not secreted. Gel filtration chromatography of rbAgg and isolated glycosaminoglycan (GAG) chains, and their susceptibility to chondroitinase ABC digestion indicate that the GAG chains are predominantly CS, which likely occupy fewer serine residues than native aggrecan. To confirm functionality, we determined that rbAgg bound hyaluronan and recombinant link protein to form proteoglycan aggregates. In addition, cleavage of rbAgg by ADAMTS-4 revealed that the p68 form of ADAMTS-4 preferentially cleaves within the CS-2 domain, whereas the p40 form only effectively cleaves within the interglobular domain (IGD). MMP-13 cleaved rbAgg within the IGD, but cleaved more rapidly at a site within the CS domains, suggesting a role in C-terminal processing of aggrecan. Our results demonstrate that recombinant aggrecan can be used for in vitro analyses of matrix protease-dependent degradation of aggrecan in the IGD and CS domains, and both recombinant aggrecan and link protein can be used to study the assembly of proteoglycan aggregates with hyaluronan.     

Investigation of the H-mode during ECRH in the T-10 tokamak

An improved confinement regime with an external transport barrier (H-mode) is obtained during electron-cyclotron resonance heating of a plasma in the T-10 tokamak. A characteristic feature of this regime is a spontaneous density growth accompanied by a drop in the intensity of D_α line and an increase in β_p by a factor of ～1.6. The threshold power for the L-H transition is close to that predicted by the ITER scaling. The best characteristics of the H-mode are achieved with decreasing q _L to 2.2. It is shown that the external transport barrier arises for electrons, whereas the heat transport barrier insignificantly contributes to improved confinement.     

Temperature‐accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain

The catalytic domain of the adenyl cyclase (AC) toxin from Bordetella pertussis is activated by interaction with calmodulin (CaM), resulting in cAMP overproduction in the infected cell. In the X‐ray crystallographic structure of the complex between AC and the C terminal lobe of CaM, the toxin displays a markedly elongated shape. As for the structure of the isolated protein, experimental results support the hypothesis that more globular conformations are sampled, but information at atomic resolution is still lacking. Here, we use temperature‐accelerated molecular dynamics (TAMD) simulations to generate putative all‐atom models of globular conformations sampled by CaM‐free AC. As collective variables, we use centers of mass coordinates of groups of residues selected from the analysis of standard molecular dynamics (MD) simulations. Results show that TAMD allows extended conformational sampling and generates AC conformations that are more globular than in the complexed state. These structures are then refined via energy minimization and further unrestrained MD simulations to optimize inter‐domain packing interactions, thus resulting in the identification of a set of hydrogen bonds present in the globular conformations. Proteins 2014; 82:2483–2496. © 2014 Wiley Periodicals, Inc.