Myricetin protects pancreatic ß-cells from human islet amyloid polypeptide (hIAPP) induced cytotoxicity and restores islet function.

The aberrant misfolding and self-assembly of human islet amyloid polypeptide (hIAPP)-a hormone that is co-secreted with insulin from pancreatic ß-cells-into toxic oligomers, protofibrils and fibrils has been observed in type 2 diabetes mellitus (T2DM). The formation of these insoluble aggregates has been linked with the death and dysfunction of ß-cells. Therefore, hIAPP aggregation has been identified as a therapeutic target for T2DM management. Several natural products are now being investigated for their potential to inhibit hIAPP aggregation and/or disaggregate preformed aggregates. In this study, we attempt to identify the anti-amyloidogenic potential of Myricetin (MYR)- a polyphenolic flavanoid, commonly found in fruits (like Syzygium cumini). Our results from biophysical studies indicated that MYR supplementation inhibits hIAPP aggregation and disaggregates preformed fibrils into non-toxic species. This protection was accompanied by inhibition of oxidative stress, reduction in lipid peroxidation and the associated membrane damage and restoration of mitochondrial membrane potential in INS-1E cells. MYR supplementation also reversed the loss of functionality in hIAPP exposed pancreatic islets via restoration of glucose-stimulated insulin secretion. Molecular dynamics simulation studies suggested that MYR molecules interact with the hIAPP pentameric fibril model at the amyloidogenic core region and thus prevents aggregation and distort the fibrils.

Chandipura Virus' Oncolytic Potential in Experimentally Induced Tumor in Mice.

Chandipura virus (CHPV) is a tropical pathogen, suggesting its involvement in childhood encephalitis syndrome in India. No reports are available in adult human beings for its pathogenicity. Similarly, in adult mice, the virus does not develop pathogenesis by parenteral route except for intracranial route of infection. The virus is remarkably nonpathogenic to adult immunocompromised nude mice. In vitro in tissue culture, the CHPV infects and kills many types of cells. All of these properties could qualify the CHPV to be a candidate virus for tumor therapy. To prove this, an experimentally induced tumor in a mouse was infected with live CHPV. The results showed that intra-tumoral injection reduced the volume of tumor and increased the longevity of the mice. The study concludes that the CHPV may be a safe tumor therapy virus. More precisely, the discovery of CHPV protein with oncolytic potential may lead to the development of novel drugs/therapeutics.

Azadirachtin inhibits amyloid formation, disaggregates pre-formed fibrils and protects pancreatic ß-cells from human islet amyloid polypeptide/amylin-induced cytotoxicity.

The human islet amyloid polypeptide (hIAPP) or amylin is the major constituent of amyloidogenic aggregates found in pancreatic islets of type 2 diabetic patients that have been associated with ß-cell dysfunction and/or death associated with type 2 diabetes mellitus (T2DM). Therefore, developing and/or identifying inhibitors of hIAPP aggregation pathway and/or compound that can mediate disaggregation of preformed aggregates holds promise as a medical intervention for T2DM management. In the current study, the anti-amyloidogenic potential of Azadirachtin (AZD)-a secondary metabolite isolated from traditional medicinal plant Neem (Azadirachta indica)-was investigated by using a combination of biophysical and cellular assays. Our results indicate that AZD supplementation not only inhibits hIAPP aggregation but also disaggregates pre-existing hIAPP fibrils by forming amorphous aggregates that are non-toxic to pancreatic ß-cells. Furthermore, AZD supplementation in pancreatic ß-cells (INS-1E) resulted in inhibition of oxidative stress; along with restoration of the DNA damage, lipid peroxidation and the associated membrane damage, endoplasmic reticulum stress and mitochondrial membrane potential. AZD treatment also restored glucose-stimulated insulin secretion from pancreatic islets exposed to hIAPP. All-atom molecular dynamics simulation studies on full-length hIAPP pentamer with AZD suggested that AZD interacted with four possible binding sites in the amyloidogenic region of hIAPP. In summary, our results suggest AZD to be a promising candidate for combating T2DM and related amyloidogenic disorders.

Conformational flexibility of histone variant CENP-ACse4 is regulated by histone H4: A mechanism to stabilize soluble Cse4.

The histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly.

Recombinant human islet amyloid polypeptide forms shorter fibrils and mediates ß-cell apoptosis via generation of oxidative stress.

Protein misfolding and aggregation play an important role in many human diseases including Alzheimer's, Parkinson's and type 2 diabetes mellitus (T2DM). The human islet amyloid polypeptide (hIAPP) forms amyloid plaques in the pancreas of T2DM subjects (>95%) that are involved in deteriorating islet function and in mediating ß-cell apoptosis. However, the detailed mechanism of action, structure and nature of toxic hIAPP species responsible for this effect remains elusive to date mainly due to the high cost associated with the chemical synthesis of pure peptide required for these studies. In the present work, we attempted to obtain structural and mechanistic insights into the hIAPP aggregation process using recombinant hIAPP (rhIAPP) isolated from Escherichia coli Results from biophysical and structural studies indicate that the rhIAPP self-assembled into highly pure, ß-sheet-rich amyloid fibrils with uniform morphology. rhIAPP-mediated apoptosis in INS-1E cells was associated with increased oxidative stress and changes in mitochondrial membrane potential. The transcript levels of apoptotic genes - Caspase-3 and Bax were found to be up-regulated, while the levels of the anti-apoptotic gene - Bcl2 were down-regulated in rhIAPP-treated cells. Additionally, the expression levels of genes involved in combating oxidative stress namely Catalase, SOD1 and GPx were down-regulated. rhIAPP exposure also affected glucose-stimulated insulin secretion from isolated pancreatic islets. The aggregation of rhIAPP also occurred significantly faster when compared with that of the chemically synthesized peptide. We also show that the rhIAPP fibrils were shorter and more cytotoxic. In summary, our study is one among the few to provide comprehensive evaluation of structural, biophysical and cytotoxic properties of rhIAPP.

Resonance assignment of disordered protein with repetitive and overlapping sequence using combinatorial approach reveals initial structural propensities and local restrictions in the denatured state.

NMR resonance assignment of intrinsically disordered proteins poses a challenge because of the limited dispersion of amide proton chemical shifts. This becomes even more complex with the increase in the size of the system. Residue specific selective labeling/unlabeling experiments have been used to resolve the overlap, but require multiple sample preparations. Here, we demonstrate an assignment strategy requiring only a single sample of uniformly labeled (13)C,(15)N-protein. We have used a combinatorial approach, involving 3D-HNN, CC(CO)NH and 2D-MUSIC, which allowed us to assign a denatured centromeric protein Cse4 of 229 residues. Further, we show that even the less sensitive experiments, when used in an efficient manner can lead to the complete assignment of a complex system without the use of specialized probes in a relatively short time frame. The assignment of the amino acids discloses the presence of local structural propensities even in the denatured state accompanied by restricted motion in certain regions that provides insights into the early folding events of the protein.

Levels of Myeloperoxidase and Alkaline Phosphatase in Periimplant Sulcus Fluid in Health and Disease and After Nonsurgical Therapy.

PURPOSE: The study evaluated 2 biomarkers (myeloperoxidase [MPO] and alkaline phosphatase) along with clinical parameters in periimplant sulcus fluid (PISF) in conditions of periimplant health and disease. The effect of nonsurgical therapy was also evaluated on the biochemical and clinical parameters in diseased implants at 3 months after the baseline. MATERIALS AND METHODS: A total number of 30 implants were studied in 20 subjects and divided into healthy group (group 1) and periimplant disease group (group 2). PISF was collected in both groups along with recording of clinical parameters. The periimplant disease group was then instituted a nonsurgical anti-infective therapy. The clinical and biochemical parameters were evaluated and compared at baseline and at the end of 3 months. RESULTS: Group 2 showed statistically significant higher PISF MPO values at baseline as compared with groups 1 and 2 (at 3 months) (P < 0.001). Also, difference in the mean value of PISF MPO in group 2 (at 3 months) was greater than that in group 1 and was statistically nonsignificant (P = 1.85). CONCLUSION: Within confines of the study, it can be concluded that evaluation of biochemical markers in PISF can be a useful diagnostic tool to aid the clinician in decision making regarding the management of the condition.

Disorder in CENP-ACse4 tail-chaperone interaction facilitates binding with Ame1/Okp1 at the kinetochore.

The centromere is epigenetically marked by a histone H3 variant-CENP-A. The budding yeast CENP-A called Cse4, consists of an unusually long N-terminus that is known to be involved in kinetochore assembly. Its disordered chaperone, Scm3 is responsible for the centromeric deposition of Cse4 as well as in the maintenance of a segregation-competent kinetochore. In this study, we show that the Cse4 N-terminus is intrinsically disordered and interacts with Scm3 at multiple sites, and the complex does not gain any substantial structure. Additionally, the complex forms a synergistic association with an essential inner kinetochore component (Ctf19-Mcm21-Okp1-Ame1), and a model has been suggested to this effect. Thus, our study provides mechanistic insights into the Cse4 N-terminus-chaperone interaction and also illustrates how intrinsically disordered proteins mediate assembly of complex multiprotein networks, in general.

Strategies for identifying dynamic regions in protein complexes: Flexibility changes accompany methylation in chemotaxis receptor signaling states.

Bacterial chemoreceptors are organized in arrays composed of helical receptors arranged as trimers of dimers, coupled to a histidine kinase CheA and a coupling protein CheW. Ligand binding to the external domain inhibits the kinase activity, leading to a change in the swimming behavior. Adaptation to an ongoing stimulus involves reversible methylation and demethylation of specific glutamate residues. However, the exact mechanism of signal propagation through the helical receptor to the histidine kinase remains elusive. Dynamics of the receptor cytoplasmic domain is thought to play an important role in the signal transduction, and current models propose inverse dynamic changes in different regions of the receptor. We hypothesize that the adaptational modification (methylation) controls the dynamics by stabilizing a partially ordered domain, which in turn modulates the binding of the kinase, CheA. We investigated the difference in dynamics between the methylated and unmethylated states of the chemoreceptor using solid-state NMR. The unmethylated receptor (CF4E) shows increased flexibility relative to the methylated mimic (CF4Q). Methylation helix 1 (MH1) has been shown to be flexible in the methylated mimic receptor. Our analysis indicates that in addition to MH1, methylation helix 2 also becomes flexible in the unmethylated receptor. In addition, we have demonstrated that both states of the receptor have a rigid region and segments with intermediate timescale dynamics. The strategies used in this study for identifying dynamic regions are applicable to a broad class of proteins and protein complexes with intrinsic disorder and dynamics spanning multiple timescales.

Carbon-nitrogen REDOR to identify ms-timescale mobility in proteins.

Protein dynamics play key mechanistic roles but are difficult to measure in large proteins and protein complexes. INEPT and CP solid-state NMR experiments have often been used to obtain spectra of protein regions that are mobile and rigid, respectively, on the nanosecond timescale. To complement this approach, we have implemented 13C{15N} REDOR to detect protein regions with backbone dynamics on the millisecond time scale that average the ≈1 kHz carbon-nitrogen dipolar coupling. REDOR-filtering of carbon correlation spectra removes signals from rigid backbone carbons and retains signals from backbone carbons with ms-timescale dynamics that would be missing in dipolar-driven NCA/NCO spectra. We use these experiments to investigate functionally important dynamics within the E coli Asp receptor cytoplasmic fragment (U-13C, 15N-CF) in native-like complexes with CheA and CheW. The CF backbone carbons exhibit only 60-75% of the expected REDOR dephasing, suggesting that 40-25% of the backbone experiences significant mobility that averages the 13C15N dipolar couplings to zero. Furthermore, the extent of this mobility changes with signaling state.