Molecular dynamics of mesophilic-like mutants of a cold-adapted enzyme: insights into distal effects induced by the mutations

Networks and clusters of intramolecular interactions, as well as their "communication" across the three-dimensional architecture have a prominent role in determining protein stability and function. Special attention has been dedicated to their role in thermal adaptation. In the present contribution, seven previously experimentally characterized mutants of a cold-adapted α-amylase, featuring mesophilic-like behavior, have been investigated by multiple molecular dynamics simulations, essential dynamics and analyses of correlated motions and electrostatic interactions. Our data elucidate the molecular mechanisms underlying the ability of single and multiple mutations to globally modulate dynamic properties of the cold-adapted α-amylase, including both local and complex unpredictable distal effects. Our investigation also shows, in agreement with the experimental data, that the conversion of the cold-adapted enzyme in a warm-adapted variant cannot be completely achieved by the introduction of few mutations, also providing the rationale behind these effects. Moreover, pivotal residues, which are likely to mediate the effects induced by the mutations, have been identified from our analyses, as well as a group of suitable candidates for protein engineering. In fact, a subset of residues here identified (as an isoleucine, or networks of mesophilic-like salt bridges in the proximity of the catalytic site) should be considered, in experimental studies, to get a more efficient modification of the features of the cold-adapted enzyme.     

New insights into the allergenicity of tropomyosin: a bioinformatics approach

The invertebrate panallergen tropomyosin is a protein with an extremely simple folding. This makes it a perfect target for investigating structural differences between invertebrate and vertebrate tropomyosins, which are not considered allergenic. Phylogenetic and sequence analyses were conducted in order to explore the differences in primary structure between several tropomyosins and to promote an experimental development in the field of food allergy, based on the study of tropomyosin. The phylogenetic analyses showed that tropomyosin is a useful evolutionary marker. The phylogenetic trees obtained with tropomyosin were not always phylogenetically correct, but they might be useful for allergen avoidance by tropomyosin allergic individuals. Sequence analyses revealed that the probability of alpha helix folding in invertebrate tropomyosins was lower than in all the studied vertebrate ones, except for the Atlantic bluefin tuna Thunnus thynnus tropomyosin. This suggested that the lack of alpha helix folding may be involved in the immunogenicity of tropomyosins. More specifically, the regions adjacent to the positions 133–135 and 201 of the invertebrate tropomyosins, presented lower probability of alpha helix folding than those of vertebrates and are candidates to be responsible for their allergenicity.     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

New molecular insights into CETP structure and function: a review

Cholesteryl ester transfer protein (CETP) is important clinically and is the current target for new drug development. Its structure and mechanism of action has not been well understood. We have combined current new structural and functional methods to compare with relevant prior data. These analyses have led us to propose several steps in CETP's function at the molecular level, in the context of its interactions with lipoproteins, e.g., sensing, penetration, docking, selectivity, ternary complex formation, lipid transfer, and HDL dissociation. These new molecular insights improve our understanding of CETP's mechanisms of action.     

Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P

In bacteria, the tRNA-processing endonuclease ribonuclease P is composed of a large ( approximately 400 nucleotide) catalytic RNA and a smaller ( approximately 100 amino acid) protein subunit that is essential for substrate recognition. Current biochemical and biophysical investigations are providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate specificity and the role of essential metal ions in catalysis. Together with recent high-resolution structures of portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and protein are coordinated in this ribonucleoprotein enzyme.     

New insights into the structure and function of potassium channels.

Potassium channels are surprisingly modular proteins. Well-defined regions that determine functional properties such as ion conduction and gating have recently been identified.     

Echoes from the past: New insights into the early hominin cochlea from a phylo-morphometric approach

We investigate cochlear variation, an indirect evidence of auditory capacities among early hominins and extant catarrhine species, in order to assess (i) the phylogenetic signal of relative external cochlear length (RECL) and oval window area (OWA), (ii) the evolutionary model with the highest probability of explaining our observed data, (iii) some hominin ancestral nodes for RECL and OWA. RECL has a high phylogenetic signal under a Brownian motion model, and is closely correlated with body mass. Our model-based method has the advantage over parsimony-based methods of incorporating branch lengths in a phylo-morphospace, and this shows RECL shifted towards significantly higher values at the Homo erectus-Homo sapiens node. We also observe that the StW 53 and KB 6067 fossil specimens from Sterkfontein and Kromdraai likely represent one or two distinct, smaller-bodied and less derived hominin form(s) compared to Paranthropus specimens represented at Swartkrans.     

New insights into fish swimming: a proteomic and isotopic approach in gilthead sea bream

Moderate exercise enhances fish growth, although underlying physiological mechanisms are not fully known. Here we performed a proteomic and metabolic study in white (WM) and red (RM) muscle of gilthead sea bream juveniles swimming at 1.5 body lengths per second. Continuous swimming for four weeks enhanced fish growth without increasing food intake. Exercise affected muscle energy stores by decreasing lipid and glycogen contents in WM and RM, respectively. Protein synthesis capacity (RNA/protein), energy use (estimated by lipid-δ(13)C and glycogen-δ(13)C), and enzymatic aerobic capacity increased in WM, while protein turnover (expressed by δ(15)N-fractionation) did not change. RM showed no changes in any of these parameters. 2D-PAGE analysis showed that almost 15% of sarcoplasmic protein spots from WM and RM differed in response to exercise, most being over-expressed in WM and under-expressed in RM. Protein identification by MALDI-TOF/TOF-MS and LC-MS/MS revealed exercise-induced enhancement of several pathways in WM (carbohydrate catabolism, protein synthesis, muscle contraction, and detoxification) and under-expression of others in RM (energy production, muscle contraction, and homeostatic processes). The mechanism underpinning the phenotypic response to exercise sheds light on the adaptive processes of fish muscles, being the sustained-moderate swimming induced in gilthead sea bream achieved mainly by WM, thus reducing the work load of RM and improving swimming performance and food conversion efficiency.     

New insights into sodium transport regulation in the distal nephron:Role of G-protein coupled receptors

The renal handling of Na~+ balance is a major determinant of the blood pressure(BP) level. The inability of the kidney to excrete the daily load of Na+ represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part(i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na~+ excretion and are the target of many different regulatory processes that modulate Na~+ retention more or less efficiently. G-protein coupled receptors(GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na~+ absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na~+ excretion, this review also highlights the complexity of these different pathways, and the connections between them.     

New insights into autophagic cell death in the gypsy moth Lymantria dispar: a proteomic approach

Autophagy is an evolutionary ancient process based on the activity of genes conserved from yeast to metazoan taxa. Whereas its role as a mechanism to provide energy during cell starvation is commonly accepted, debate continues about the occurrence of autophagy as a means specifically activated to achieve cell death. The IPLB-LdFB insect cell line, derived from the larval fat body of the lepidoptera Lymantria dispar, represents a suitable model to address this question, as both autophagic and apoptotic cell death can be induced by various stimuli. Using morphological and functional approaches, we have observed that the culture medium conditioned by IPLB-LdFB cells committed to death by the ATPase inhibitor oligomycin A stimulates autophagic cell death in untreated IPLB-LdFB cells. Moreover, proteomic analysis of the conditioned media suggests that, in IPLB-LdFB cells, oligomycin A promotes a shift towards lipid metabolism, increases oxidative stress and specifically directs the cells towards autophagic activity. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by an F.A.R. grant from the University of Modena and Reggio Emilia (D.M. and E.O.) and by an “Experimental approaches to the study of evolution” grant from the Department of Animal Biology of the University of Modena and Reggio Emilia (D.M.).     

Computational insights into pH-dependence of structure and dynamics of pyrazinamidase: A comparison of wild type and mutants

The mycobacterial enzyme pyrazinamidase (PZase) is the target of key tuberculosis drug, pyrazinamide. Mutations in PZase cause drug resistance. Herein, three point mutations, W68G, L85P, and V155G, were investigated through over 8 µs of molecular dynamics simulations coupled with essential dynamics and binding pocket analysis at neutral (pH = 7) and acidic (pH = 4) ambient conditions. The 51-71 flap region exhibited drastic displacement leading to enlargement of binding cavity, especially at the lower pH. Accessibility of solvent to the active site of the mutant enzymes was also reduced. The protonation of key surface residues at low pH results in more contribution of these residues to structural stability and integrity of the enzyme and reduced interactions with solvent molecules, which acts as a cage, keeping the enzyme together. The observed results suggest a pattern of structural alterations due to point mutations in PZase, which is consistent with other experimental and theoretical investigations and, can be harnessed for drug design purposes.     

Expression and characterization of cyanobacterium heme oxygenase, a key enzyme in the phycobilin synthesis. Properties of the heme complex of recombinant active enzyme.

An efficient bacterial expression system of cyanobacterium Synechocystis sp. PCC 6803 heme oxygenase gene, ho-1, has been constructed, using a synthetic gene. A soluble protein was expressed at high levels and was highly purified, for the first time. The protein binds equimolar free hemin to catabolize the bound hemin to ferric-biliverdin IX alpha in the presence of oxygen and reducing equivalents, showing the heme oxygenase activity. During the reaction, verdoheme intermediate is formed with the evolution of carbon monoxide. Though both ascorbate and NADPH-cytochrome P450 reductase serve as an electron donor, the heme catabolism assisted by ascorbate is considerably slow and the reaction with NADPH-cytochrome P450 reductase is greatly retarded after the oxy-heme complex formation. The optical absorption spectra of the heme-enzyme complexes are similar to those of the known heme oxygenase complexes but have some distinct features, exhibiting the Soret band slightly blue-shifted and relatively strong CT bands of the high-spin component in the ferric form spectrum. The heme-enzyme complex shows the acid-base transition, where two alkaline species are generated. EPR of the nitrosyl heme complex has established the nitrogenous proximal ligand, presumably histidine 17 and the obtained EPR parameters are discriminated from those of the rat heme oxygenase-1 complex. The spectroscopic characters as well as the catabolic activities strongly suggest that, in spite of very high conservation of the primary structure, the heme pocket structure of Synechocystis heme oxygenase isoform-1 is different from that of rat heme oxygenase isoform-1, rather resembling that of bacterial heme oxygenase, H mu O.     

Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29.     

New insights into the mechanisms of embryonic stem cell self-renewal under hypoxia: a multifactorial analysis approach

Previous reports have shown that culturing mouse embryonic stem (mES) cells at different oxygen tensions originated different cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support the pluripotency state. Herein we provide new insights into the mechanisms by which oxygen is influencing mES cell self-renewal and pluripotency. A multifactorial approach was developed to rationally evaluate the singular and interactive control of MEK/ERK pathway, GSK-3 inhibition, and LIF/STAT3 signaling at physiological and non-physiological oxygen tensions. Collectively, our methodology revealed a significant role of GSK-3-mediated signaling towards maintenance of mES cell pluripotency at lower O(2) tensions. Given the central role of this signaling pathway, future studies will need to focus on the downstream mechanisms involved in ES cell self-renewal under such conditions, and ultimately how these findings impact human models of pluripotency.