Identification of a new cross-link and unique histidine adduct from bovine serum albumin incubated with malondialdehyde

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.     

X-ray crystal structure of the hypothetical phosphotyrosine phosphatase MDP-1 of the haloacid dehalogenase superfamily

The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 A X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Disruption of Caenorhabditis elegans muscle structure and function caused by mutation of troponin I

Caenorhabditis elegans strains mutant for the unc-27 gene show abnormal locomotion and muscle structure. Experiments revealed that unc-27 is one of four C. elegans troponin I genes and that three mutant alleles truncate the protein: recessive and presumed null allele e155 terminates after nine codons; semidominant su142sd eliminates the inhibitory and C-terminal regions; and semidominant su195sd abbreviates the extreme C-terminus. Assays of in vivo muscular performance at high and low loads indicated that su142sd is most deleterious, with e155 least and su195sd intermediate. Microscopy revealed in mutant muscle a prevalent disorder of dense body positioning and a less well defined sarcomeric structure, with small islands of thin filaments interspersed within the overlap region of A bands and even within the H zone. The mutants' rigid paralysis and sarcomeric disarray are consistent with unregulated contraction of the sarcomeres, in which small portions of each myofibril shorten irregularly and independently of one another, thereby distorting the disposition of filaments. The exacerbated deficits of su142sd worms are compatible with involvement in vivo of the N-terminal portion of troponin I in enhancing force production, and the severe impairment associated with su195sd highlights importance of the extreme C-terminus in the protein's inhibitory function.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.     

Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population

Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited. Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens. The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S. suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds. Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell. Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene. Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates. In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide. There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S. suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia.     

Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C delta-dependent pathway

Myosin-based cell contractile force is considered to be a critical process in cell motility. However, for epidermal growth factor (EGF)-induced fibroblast migration, molecular links between EGF receptor (EGFR) activation and force generation have not been clarified. Herein, we demonstrate that EGF stimulation increases myosin light chain (MLC) phosphorylation, a marker for contractile force, concomitant with protein kinase C (PKC) activity in mouse fibroblasts expressing human EGFR constructs. Interestingly, PKCdelta is the most strongly phosphorylated isoform, and the preferential PKCdelta inhibitor rottlerin largely prevented EGF-induced phosphorylation of PKC substrates and MARCKS. The pathway through which EGFR activates PKCdelta is suggested by the fact that the MEK-1 inhibitor U0126 and the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on PKCdelta activation, whereas lack of PLCgamma signaling resulted in delayed PKCdelta activation. EGF-enhanced MLC phosphorylation was prevented by a specific MLC kinase inhibitor ML-7 and the PKC inhibitors chelerythrine chloride and rottlerin. Further indicating that PKCdelta is required, a dominant-negative PKCdelta construct or RNAi-mediated PKCdelta depletion also prevented MLC phosphorylation. In the absence of PLC signaling, MLC phosphorylation and cell force generation were delayed similarly to PKCdelta activation. All of the interventions that blocked PKCdelta activation or MLC phosphorylation abrogated EGF-induced cell contractile force generation and motility. Our results suggest that PKCdelta activation is responsible for a major part of EGF-induced fibroblast contractile force generation. Hence, we identify here a new pathway helping to govern cell motility, with PLC signaling playing a role in activation of PKCdelta to promote the acute phase of EGF-induced MLC activation.     

DNA interaction and dimerization of eukaryotic SMC hinge domains

The eukaryotic SMC1/SMC3 heterodimer is essential for sister chromatid cohesion and acts in DNA repair and recombination. Dimerization depends on the central hinge domain present in all SMC proteins, which is flanked at each side by extended coiled-coil regions that terminate in specific globular domains. Here we report on DNA interactions of the eukaryotic, heterodimeric SMC1/SMC3 hinge regions, using the two known isoforms, SMC1alpha/SMC3 and the meiotic SMC1beta/SMC3. Both dimers bind DNA with a preference for double-stranded DNA and DNA rich in potential secondary structures. Both dimers form large protein-DNA networks and promote reannealing of complementary DNA strands. DNA binding but not dimerization depends on approximately 20 amino acids of transitional sequence into the coiled-coil region. Replacement of three highly conserved glycine residues, thought to be required for dimerization, in one of the two hinge domains still allows formation of a stable dimer, but if two hinge domains are mutated dimerization fails. Single-mutant dimers bind DNA, but hinge monomers do not. Together, we show that eukaryotic hinge dimerization does not require conserved glycines in both hinge domains, that only the transition into the coiled-coil region rather than the entire coiled-coil region is necessary for DNA binding, and that dimerization is required but not sufficient for DNA binding of the eukaryotic hinge heterodimer.