Protein contact prediction using patterns of correlation

We describe a new method for using neural networks to predict residue contact pairs in a protein. The main inputs to the neural network are a set of 25 measures of correlated mutation between all pairs of residues in two "windows" of size 5 centered on the residues of interest. While the individual pair-wise correlations are a relatively weak predictor of contact, by training the network on windows of correlation the accuracy of prediction is significantly improved. The neural network is trained on a set of 100 proteins and then tested on a disjoint set of 1033 proteins of known structure. An average predictive accuracy of 21.7% is obtained taking the best L/2 predictions for each protein, where L is the sequence length. Taking the best L/10 predictions gives an average accuracy of 30.7%. The predictor is also tested on a set of 59 proteins from the CASP5 experiment. The accuracy is found to be relatively consistent across different sequence lengths, but to vary widely according to the secondary structure. Predictive accuracy is also found to improve by using multiple sequence alignments containing many sequences to calculate the correlations.     

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.     

A multi-scaled approach for simulating chemical reaction systems

In this paper we give an overview of some very recent work, as well as presenting a new approach, on the stochastic simulation of multi-scaled systems involving chemical reactions. In many biological systems (such as genetic regulation and cellular dynamics) there is a mix between small numbers of key regulatory proteins, and medium and large numbers of molecules. In addition, it is important to be able to follow the trajectories of individual molecules by taking proper account of the randomness inherent in such a system. We describe different types of simulation techniques (including the stochastic simulation algorithm, Poisson Runge–Kutta methods and the balanced Euler method) for treating simulations in the three different reaction regimes: slow, medium and fast. We then review some recent techniques on the treatment of coupled slow and fast reactions for stochastic chemical kinetics and present a new approach which couples the three regimes mentioned above. We then apply this approach to a biologically inspired problem involving the expression and activity of LacZ and LacY proteins in E. coli, and conclude with a discussion on the significance of this work.     

Molecular epidemiology of nosocomial infection by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae

Molecular epidemiology applied to the study of nosocomial infection has been fundamental in formulating and evaluating control methods. From patients in a level 3 Bogota hospital, Klebsiella pneumoniae samples were isolated that produced extended-spectrum beta-lactamases (ESBL). Each of 15 isolates was characterized microbiologically and by molecular characters realized by pulsed field gel electrophoresis (PFGE) and by repetitive-DNA sequences amplification (REP-PCR). Antimicrobial susceptibility and ESBL production was determined in accordance with NCCLS guidelines. The beta-lactamases were evaluated by isoelectric-focusing and PCR. Twelve (80%) of the isolates were associated with nosocomial infection; 11 of them were from intensive care units. The antibiotic susceptibility displayed 13 resistance patterns--87% presented co-resistance to amikacin, 53% to gentamicin, 33% to ciprofloxacin, 40% to cefepime, 67% to piperacillin/tazobactam, 60% to trimethoprim/sulfamethoxazole and 47% to chloranphenicol. All were sensitive to imipenem. Production of TEM and SHV beta-lactamases was detected simultaneously in most isolates by isoelectric focusing and 93.3% produced a ceftazidimase of pl 8.2 of the SHV-5 type. The 15 isolates were grouped into 11 and 12 electrophoretic patterns by PFGE and REP-PCR, respectively. The degree of genetic variability indicated an endogenous origin of the nosocomial infections.     

In vivo incorporation of selenomethionine in proteins using Pseudomonas aeruginosa as expression host: case study-the outer membrane receptor FpvA

The number of protein structures solved using multiwavelength anomalous diffraction methods coupled with selenomethionine substitution has grown dramatically over the last years. We show using the outer membrane pyoverdin receptor FpvA that Pseudomonas aeruginosa can be used for producing proteins with a high level of selenomethionine incorporation. To circumvent problems encountered with mass spectroscopy analysis of purified membrane proteins, in-gel trypsin digestion of FpvA coupled with MALDI mass spectrometry analysis of the resulting peptides was used to determine the extent of selenomethionine incorporation. Selenomethionine incorporation greater than 95% was achieved using P. aeruginosa as an overexpression system.     

A model system for increased meiotic nondisjunction in older oocytes

For at least 5% of all clinically recognized human pregnancies, meiotic segregation errors give rise to zygotes with the wrong number of chromosomes. Although most aneuploid fetuses perish in utero, trisomy in liveborns is the leading cause of mental retardation. A large percentage of human trisomies originate from segregation errors during female meiosis I; such errors increase in frequency with maternal age. Despite the clinical importance of age-dependent nondisjunction in humans, the underlying mechanisms remain largely unexplained. Efforts to recapitulate age-dependent nondisjunction in a mammalian experimental system have so far been unsuccessful. Here we provide evidence that Drosophila is an excellent model organism for investigating how oocyte aging contributes to meiotic nondisjunction. As in human oocytes, nonexchange homologs and bivalents with a single distal crossover in Drosophila oocytes are most susceptible to spontaneous nondisjunction during meiosis I. We show that in a sensitized genetic background in which sister chromatid cohesion is compromised, nonrecombinant X chromosomes become vulnerable to meiotic nondisjunction as Drosophila oocytes age. Our data indicate that the backup pathway that normally ensures proper segregation of achiasmate chromosomes deteriorates as Drosophila oocytes age and provide an intriguing paradigm for certain classes of age-dependent meiotic nondisjunction in humans.     

Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments

Although many human immunodeficiency virus type 1 (HIV-1)-infected persons are treated with multiple protease inhibitors in combination or in succession, mutation patterns of protease isolates from these persons have not been characterized. We collected and analyzed 2,244 subtype B HIV-1 isolates from 1,919 persons with different protease inhibitor experiences: 1,004 isolates from untreated persons, 637 isolates from persons who received one protease inhibitor, and 603 isolates from persons receiving two or more protease inhibitors. The median number of protease mutations per isolate increased from 4 in untreated persons to 12 in persons who had received four or more protease inhibitors. Mutations at 45 of the 99 amino acid positions in the protease-including 22 not previously associated with drug resistance-were significantly associated with protease inhibitor treatment. Mutations at 17 of the remaining 99 positions were polymorphic but not associated with drug treatment. Pairs and clusters of correlated (covarying) mutations were significantly more likely to occur in treated than in untreated persons: 115 versus 23 pairs and 30 versus 2 clusters, respectively. Of the 115 statistically significant pairs of covarying residues in the treated isolates, 59 were within 8 A of each other-many more than would be expected by chance. In summary, nearly one-half of HIV-1 protease positions are under selective drug pressure, including many residues not previously associated with drug resistance. Structural factors appear to be responsible for the high frequency of covariation among many of the protease residues. The presence of mutational clusters provides insight into the complex mutational patterns required for HIV-1 protease inhibitor resistance.     

Viability of a drug-resistant human immunodeficiency virus type 1 protease variant: structural insights for better antiviral therapy

Under the selective pressure of protease inhibitor therapy, patients infected with human immunodeficiency virus (HIV) often develop drug-resistant HIV strains. One of the first drug-resistant mutations to arise in the protease, particularly in patients receiving indinavir or ritonavir treatment, is V82A, which compromises the binding of these and other inhibitors but allows the virus to remain viable. To probe this drug resistance, we solved the crystal structures of three natural substrates and two commercial drugs in complex with an inactive drug-resistant mutant (D25N/V82A) HIV-1 protease. Through structural analysis and comparison of the protein-ligand interactions, we found that Val82 interacts more closely with the drugs than with the natural substrate peptides. The V82A mutation compromises these interactions with the drugs while not greatly affecting the substrate interactions, which is consistent with previously published kinetic data. Coupled with our earlier observations, these findings suggest that future inhibitor design may reduce the probability of the appearance of drug-resistant mutations by targeting residues that are essential for substrate recognition.     

Assembly and molecular activities of the MutS tetramer

Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 x 10(7) M-1, indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. (2000) Nature 407, 703-710; Lamers, M. H., Perrakis, A., Enzlin, J. H., Winterwerp, H. H., de Wind, N., and Sixma, T. K. (2000) Nature 407, 711-717). MutS800 binds a 20-base pair heteroduplex an order of magnitude more weakly than full-length MutS, and at saturating protein concentrations, the heteroduplex-bound mass observed with MutS800 is only half that observed with the full length protein, indicating that the subunit copy number of heteroduplex-bound MutS is twice that of MutS800. Analytical equilibrium ultracentrifugation using a fluorescein-tagged 20-base pair heteroduplex demonstrated that native MutS forms a tetramer on this single site-sized heteroduplex DNA. Equilibrium fluorescence experiments indicated that dimer-to-tetramer assembly promotes mismatch binding by MutS and that the tetramer can bind only a single heteroduplex molecule, implying nonequivalence of the two dimers within the tetramer. Compared with native MutS, the ability of MutS800 to promote MutL-dependent activation of MutH is substantially reduced.