Impact of non-synonymous mutations on the structure and function of telomeric repeat binding factor 1.

Telomeric repeat binding factor 1 (TRF1) is one of the major components of the shelterin complex. It directly binds to the telomere and controls its function by regulating the telomerase acting on it. Several variations are reported in the TRF1 gene; some are associated with variety of diseases. Here, we have studied the structural and functional significance of these variations in the TRFH domain of TRF1. We have used cutting-edge computational methods such as SIFT, PolyPhen-2, PROVEAN, Mutation Assessor, mCSM, SDM, STRUM, MAESTRO, and DUET to predict the effects of 124 mutations in the TRFH domain of TRF1. Out of 124 mutations, we have identified 12 deleterious mutations with high confidence based on their prediction. To see the impact of the finally selected mutations on the structure and stability of TRF1, all-atom molecular dynamics (MD) simulations on TRF1-Wild type (WT), L79R and P150R mutants for 200 ns were carried out. A significant conformational change in the structure of the P150R mutant was observed. Our integrated computational study provides a comprehensive understanding of structural changes in TRF1 incurred due to the mutations and subsequent function, leading to the progression of many diseases.Communicated by Ramaswamy H. Sarma.

Non-enzymatic glycation of protein induces cancer cell proliferation and its inhibition by quercetin: Spectroscopic, cytotoxicity and molecular docking studies.

Methylglyoxal (MG) is a potent glycating agent which reacts with proteins to form advanced glycation end products (AGEs). These chemically stable AGEs crosslink with proteins and could lead to amyloid formation that has the role in several diseases including Alzheimer's and Parkinson's. In this piece of work, glycation-induced conformational changes in HSA were observed with quenching of tryptophan fluorescence by 73.8% (41 nm red shift) and loss of hydrophobicity of HSA. CD spectroscopy result reaffirmed secondary structure changes in HSA. Moreover, MG-induced changes in HSA, proceeds to amyloid structure as characterized by an increase in thioflavin (ThT) fluorescence and transmission electron microscopy (TEM) images of HSA aggregates. Quercetin was found to inhibit both AGEs production and amyloid formation. Viability of MCF-7 cells was found to be increased with AGEs treatment, illustrating proliferation of cancer cells. Wound healing assay also revealed increased proliferation and migration of cells in the presence of AGEs. Additionally, molecular docking analyses were performed to demonstrate interactions involved in the stabilization of HSA-quercetin complex. The binding affinities of quercetin were found to be (K d = 105 M -1) much higher compared with MG (K d = 102 M -1). From this study, it is quite clear that quercetin reverses the effect of MG by sterically inhibiting the interaction between HSA and MG. Communicated by Ramaswamy H. Sarma.

Impact of Gln94Glu mutation on the structure and function of protection of telomere 1, a cause of cutaneous familial melanoma.

Protection of telomere 1 (POT1) is a key component of shelterin complex, essential for maintaining telomere length and its regulation. It consists of N-terminal domain (residues 1-299), which interacts with telomeric ssDNA, and the C-terminal domain (residues 320-634) that binds to the tripeptidyl-peptidase I (TPP1). A large number of naturally occurring mutations in the POT1 gene are associated with glioma, cardiac angiosarcoma and cutaneous familial melanoma (FM). In particular, Q94E mutation disrupts the interaction of POT1 with telomeric DNA which subsequently enhances telomere uncapping and elongation and promotes the development of cutaneous familial melanoma. To understand the underlying mechanism of familial melanoma developed by Q94E-mutation, we have performed extensive structure analysis of WT and mutant protein followed by molecular dynamics simulations. Q94E mutation causes a dramatic change in the structure and stability of POT1 protein. A considerable decrease in the flexibility, fluctuation and solvent accessibility of Q94E was observed in comparison to the WT, indicating overall destabilization of protein. Essential dynamics and Anisotropic Network Mode analysis have quantified a significant change in direction and magnitude of conformational motion in Q94E mutant compared to WT. A significant loss of frustration due to Q94E mutation was also observed. Our findings indicate the loss of protein stability and dynamics of POT1 protein by Q94E mutation may be associated with the familial melanoma. AbbreviationsANManisotropic network modeEDessential dynamicsFMfamilial melanomaMDmolecular dynamicsPOT1protection of telomere 1Rgradius of gyrationRMSDroot-mean-square deviationRMSFroot-mean-square fluctuationsSASAsolvent accessible surface areaSIFTsorting Intolerant from TolerantTPP1tripeptidyl-peptidase IWTwild typeCommunicated by Ramaswamy H. Sarma.

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor.

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both "loss as well as gain of function" mutations observed in this domain. Naturally occurring "gain of function" mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These "gain of function" mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the "gain of function" effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.

Osmolytes in vaccine production, flocculation and storage: a critical review.

Small molecule osmolytes, responsible for protecting stresses have long been known to rescue proteins and enzymes from loss of function. In addition to protecting macromolecules integrity, many osmolytes also act as potential antioxidant and also help to prevent protein aggregation, amyloid formation or misfolding, and therefore are considered promising molecules for neurodegenerative and many other genetic diseases. Osmolytes are also known to be involved in the regulation of several key immunological processes. In the present review we discuss in detail the effect of these compounds on important aspects of vaccines i.e., increasing the efficiency, production and purification steps. The present review therefore will help researchers to make a better strategy in vaccine production to formulation by incorporating specific and appropriate osmolytes in the processes.