Search results for "Mutant Protein"
showing 10 items of 42 documents
Probing a Polar Cluster in the Retinal Binding Pocket of Bacteriorhodopsin by a Chemical Design Approach
2012
Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoprotein…
Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches
2013
The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2-fold improvement in kcat, 5.2-fold lower Km and 16-fold improvement in catalytic efficiency for D-tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased kcat for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher kcat and Km value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D-tyrosine. Single muta…
Dynamics of a Protein Interaction Network Associated to the Aggregation of polyQ-Expanded Ataxin-1
2020
Background: Several experimental models of polyglutamine (polyQ) diseases have been previously developed that are useful for studying disease progression in the primarily affected central nervous system. However, there is a missing link between cellular and animal models that would indicate the molecular defects occurring in neurons and are responsible for the disease phenotype in vivo. Methods: Here, we used a computational approach to identify dysregulated pathways shared by an in vitro and an in vivo model of ATXN1(Q82) protein aggregation, the mutant protein that causes the neurodegenerative polyQ disease spinocerebellar ataxia type-1 (SCA1). Results: A set of common dysregulated pathwa…
Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.
2016
Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel find…
FUS MUTATIONS IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS: CLINICAL AND GENETIC ANALYSIS
2012
Fused in sarcoma (FUS) or translocation in liposarcoma (TLS), a DNA/RNA-binding protein, causes a dominant autosomal inherited form of amyotrophic lateral sclerosis (ALS), ALS 6. Its main role in neurodegeneration is highlighted by the presence of cytoplasmic accumulation of its mutant protein form in ALS patients. To further define the frequency and spectrum of FUS gene mutations, we have performed a molecular screening of a cohort of 327 Italian patients from Southern Italy with sporadic ALS (SALS). We identified 4 patients carrying 3 different missense mutations and several polymorphisms. Two different substitutions occurring in the same amino acidic position have been observed in 2 pati…
Structural Basis and Enzymatic Mechanism of the Biosynthesis of C9- from C10-Monoterpenoid Indole Alkaloids
2009
Cutting carbons: The three-dimensional structure of polyneuridine aldehyde esterase (PNAE) gives insight into the enzymatic mechanism of the biosynthesis of C(9)- from C(10)-monoterpenoid indole alkaloids (see scheme). PNAE is a very substrate-specific serine esterase. It harbors the catalytic triad S87-D216-H244, and is a new member of the alpha/beta-fold hydrolase superfamily. Its novel function leads to the diversification of alkaloid structures.
Tetraspanin CD151 Mediates Papillomavirus Type 16 Endocytosis
2013
ABSTRACT Human papillomavirus type 16 (HPV16) is the primary etiologic agent for cervical cancer. The infectious entry of HPV16 into cells occurs via a so-far poorly characterized clathrin- and caveolin-independent endocytic pathway, which involves tetraspanin proteins and actin. In this study, we investigated the specific role of the tetraspanin CD151 in the early steps of HPV16 infection. We show that surface-bound HPV16 moves together with CD151 within the plane of the membrane before they cointernalize into endosomes. Depletion of endogenous CD151 did not affect binding of viral particles to cells but resulted in reduction of HPV16 endocytosis. HPV16 uptake is dependent on the C-termina…
Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family[W]
2007
Abstract Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids …
Targeting Cavity-Creating p53 Cancer Mutations with Small-Molecule Stabilizers: the Y220X Paradigm
2020
We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pock…
The Histidinol Phosphate Phosphatase Involved in Histidine Biosynthetic Pathway Is Encoded by SCO5208 (hisN) in Streptomyces coelicolor A3(2)
2008
Through the screening of a Streptomyces coelicolor genomic library, carried out in a histidinol phosphate phosphatase (HolPase) deficient strain, SCO5208 was identified as the last unknown gene involved in histidine biosynthesis. SCO5208 is a phosphatase, and it can restore the growth in minimal medium in this HolPase deficient strain when cloned in a high or low copy number vector. Moreover, it shares sequence homology with other HolPases recently identified in Actinobacteria. During this work a second phosphatase, SCO2771, sharing no homologies with SCO5208 and all so far described phosphatases was identified. It can complement HolPase activity mutation only at high copy number. Sequence …