A Novel Mitochondrial Heteroplasmic C13806A Point Mutation Associated with Iranian Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by decreased expression of the protein Frataxin. Frataxin deficiency leads to excessive free radical production and dysfunction of chain complexes. Mitochondrial DNA (mtDNA) could be considered a candidate modifier factor for FRDA disease, since mitochondrial oxidative stress is thought to be involved in the pathogenesis of this disease. It prompted us to focus on the mtDNA and monitor the nucleotide changes of genome which are probably the cause of respiratory chain defects and reduced ATP generation. We searched about 46% of the entire mitochondrial genome by temporal temperature gradient gel electrophoresis (TTGE) and DNA fragments showing abnormal banding patterns were sequenced for the identification of exact mutations. In 18 patients, for the first time, we detected 26 mtDNA mutations; of which 5 (19.2%) was novel and 21 (80.8%) have been reported in other diseases. Heteroplasmic C13806A polymorphisms were associated with Iranian FRDA patients (55.5%). Our results showed that NADH dehydrogenase (ND) genes mutations in FRDA samples were higher than normal controls (P < 0.001) and we found statistically significant inverse correlation (r = −0.8) between number of mutation in ND genes and age of onset in FRDA patients. It is possible that mutations in ND genes could constitute a predisposing factor which in combination with environmental risk factors affects age of onset and disease progression.     

Mechanistic Basis for the Emergence of Catalytic Competence against Carbapenem Antibiotics by the GES Family of ��-Lactamases

A major mechanism of bacterial resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is the production of β-lactamases. A handful of class A β-lactamases have been discovered that have acquired the ability to turn over carbapenem antibiotics. This is a disconcerting development, as carbapenems are often considered last resort antibiotics in the treatment of difficult infections. The GES family of β-lactamases constitutes a group of extended spectrum resistance enzymes that hydrolyze penicillins and cephalosporins avidly. A single amino acid substitution at position 170 has expanded the breadth of activity to include carbapenems. The basis for this expansion of activity is investigated in this first report of detailed steady-state and pre-steady-state kinetics of carbapenem hydrolysis, performed with a class A carbapenemase. Monitoring the turnover of imipenem (a carbapenem) by GES-1 (Gly-170) revealed the acylation step as rate-limiting. GES-2 (Asn-170) has an enhanced rate of acylation, compared with GES-1, and no longer has a single rate-determining step. Both the acylation and deacylation steps are of equal magnitude. GES-5 (Ser-170) exhibits an enhancement of the rate constant for acylation by a remarkable 5000-fold, whereby the enzyme acylation event is no longer rate-limiting. This carbapenemase exhibits k_cat/K_m of 3 × 10⁵ m⁻¹s⁻¹, which is sufficient for manifestation of resistance against imipenem.Bacterial production of β-lactamases is a primary mechanism of resistance to β-lactam antibiotics (1). These enzymes hydrolytically process the β-lactam bond of the antibiotic, and by so doing, inactivate them. Four classes of β-lactamases, A, B, C, and D, are known, of which the class A enzymes are most prevalent (1, 2). Enzymes belonging to classes A, C, and D are serine-dependent. A critical active site serine in these enzymes experiences acylation by the antibiotic followed by deacylation. The mechanistic details of the deacylation steps vary for each class (1). Class B enzymes are zinc-dependent, and their mechanistic details are distinct. Random mutations in the genes for these enzymes have allowed for selection of novel variants within the clinic having an increased breadth for substrate preference (3). Variants of β-lactamases with the ability to turn over both penicillins and cephalosporins are referred to as extended spectrum β-lactamases (4). Extended spectrum β-lactamases typically do not have the ability to hydrolyze carbapenems; however, exceptions to this rule are emerging among members of classes A, B, and D and are called carbapenemases (5, 6). Within class A, members of the KPC and GES families are becoming increasingly problematic within the clinic (3, 5, 7). The GES type class A β-lactamases were identified for the first time only 10 years ago, but they have been increasingly detected worldwide among Gram-negative bacteria (3). The first variants detected were plasmid borne and isolated from Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Enterobacter cloacae (8–14); however, newer variants have been identified in the chromosome of P. aeruginosa (15, 16). A distinguishing characteristic of this family of enzymes is its ability to develop resistance to all classes of β-lactam antibiotics, including third-generation cephalosporins, cephamycins, monobactams, and/or carbapenems. This is in stark contrast to “classical” β-lactamases, such as the TEM family of enzymes, which also rapidly evolved to produce numerous extended spectrum and inhibitor-resistant variants, yet have failed to evolve activity against carbapenem antibiotics. This combination of resistance to both extended spectrum β-lactams and carbapenem antibiotics makes the organisms that harbor the genes for these enzymes dangerous in a clinical setting as treatment options dwindle.GES-1, the first member of the GES family to be identified, has no significant ability to hydrolyze carbapenems, leading to its classification as only an extended spectrum β-lactamase (10). Other GES-type enzymes were later discovered with increased resistance to carbapenems. Two of these variants, GES-2 and -5, contain only a single amino acid substitution compared with the sequence of GES-1, both at position 170 (Ambler numbering is used) (17). This position is located within an Ω-loop forming one of the walls of the active site (1). The canonical residue at this position of class A β-lactamases is asparagine, which is found in GES-2. However, GES-1 contains a glycine, and GES-5 contains a serine (17). We recently solved the x-ray crystal structure of the GES-1 enzyme; however, it was not clear from the structural data why such a substitution would convey resistance in GES-2 and -5, in contrast to the case of GES-1, which does not (18). The mechanistic basis for the extended profile for resistance is not known, and it is the subject of study in this report. Previous kinetic analyses of class A carbapenemases, including GES enzymes, have been limited to steady-state kinetic parameters (8, 10, 11). In this report, we have performed an in-depth analysis of the GES family, including both steady-state and the first pre-steady-state analyses (for any class A carbapenemase) to elucidate the nature of the microscopic steps (binding, acylation, deacylation, and product release) in the turnover process by these clinically important enzymes.     

Crystal structure of CbpF,a bifunctional choline‐binding protein and autolysis regulator from Streptococcus pneumoniae

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline‐binding proteins. The three‐dimensional structure of choline‐binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non‐consensus choline‐binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well‐defined modules. The carboxy‐terminal module is involved in cell wall binding and the amino‐terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.     

High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli

The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane.     

Recognition of peptidoglycan and β-lactam antibiotics by the extracellular domain of the Ser/Thr protein kinase StkP from Streptococcus pneumoniae

The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular C-terminal domain of StkP kinase (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan (PGN) and to β-lactam antibiotics, which mimic the terminal portions of the PGN stem peptide. This is the first experimental report on the recognition of a minimal PGN unit by a PASTA-containing kinase, suggesting that non-crosslinked PGN may act as a signal for StkP function and pointing to this protein as an interesting target for β-lactam antibiotics.     

Comparative molecular field analysis and QSAR on substrates binding to cytochrome p450 2D6

In this study, we utilized comparative molecular field analysis (CoMFA) to gain a better understanding of the steric and electrostatic features of the cytochrome P450 2D6 (CYP2D6) active site. The training set consists of 24 substrates with reported K_M values from liver microsomal CYP2D6 spanning an activity range of almost three log units. The low energy conformers were fit by root mean square (RMS) to minaprine at the site of metabolism and to the protonated nitrogen. In this manner, we constructed two CoMFA models, one model with a distance constraint and another without. The model with the distance parameter (non-crossvalidated R²=0.99) was approximately equal to the CoMFA without a distance parameter (non-cross-validated R²=0.98). Validation of our CoMFA was accomplished by predicting the K_M values of 15 diverse CYP2D6 substrates not in the original training set resulting in a predictive R²=0.62. Finally, we also pursued correlations of pK_a and log P with CYP2D6 substrate K_M in an effort to investigate other physicochemical properties.     

Evaluation of inhibition of the carbenicillin-hydrolyzing β-lactamase PSE-4 by the clinically used mechanism-based inhibitors

Characterization of the biochemical steps in the inactivation chemistry of clavulanic acid, sulbactam and tazobactam with the carbenicillin-hydrolyzing β-lactamase PSE-4 from Pseudomonas aeruginosa is described. Although tazobactam showed the highest affinity to the enzyme, all three inactivators were excellent inhibitors for this enzyme. Transient inhibition was observed for the three inactivators before the onset of irreversible inactivation of the enzyme. Partition ratios (k_cat/k_inact) of 11, 41 and 131 were obtained with clavulanic acid, tazobactam and sulbactam, respectively. Furthermore, these values were found to be 14-fold, 3-fold and 80-fold lower, respectively, than the values obtained for the clinically important TEM-1 β-lactamase. The kinetic findings were put in perspective by determining the computational models for the pre-acylation complexes and the immediate acyl-enzyme intermediates for all three inactivators. A discussion of the pertinent structural factors is presented, with PSE-4 showing subtle differences in interactions with the three inhibitors compared to the TEM-1 enzyme.     

In silico and in vitro effects of the I30T mutation on myelin protein zero instability in the cell membrane

Charcot‐Marie‐Tooth (CMT) diseases are a heterogeneous group of genetic peripheral neuropathies caused by mutations in a variety of genes, which are involved in the development and maintenance of peripheral nerves. Myelin protein zero (MPZ) is expressed by Schwann cells, and MPZ mutations can lead to primarily demyelinating polyneuropathies including CMT type 1B. Different mutations demonstrate various forms of disease pathomechanisms, which may be beneficial in understanding the disease cellular pathology. Our molecular dynamics simulation study on the possible impacts of I30T mutation on the MPZ protein structure suggested a higher hydrophobicity and thus lower stability in the membranous structures. A study was also conducted to predict native/mutant MPZ interactions. To validate the results of the simulation study, the native and mutant forms of the MPZ protein were separately expressed in a cellular model, and the protein trafficking was chased down in a time course pattern. In vitro studies provided more evidence on the instability of the MPZ protein due to the mutation. In this study, qualitative and quantitative approaches were adopted to confirm the instability of mutant MPZ in cellular membranes.     

Harnessing Bioinformatic Approaches to Design Novel Multi-epitope Subunit Vaccine Against Leishmania infantum

International Journal of Peptide Research and Therapeutics - Kala-azar or visceral leishmaniasis (VL) is the most important vector-borne protozoan disease and a life-threatening problem in the...