Specificity of Streptococcus pyogenes NAD(+) glycohydrolase in cytolysin-mediated translocation

The mechanism by which the cytolysin-mediated translocation (CMT) pathway of the Gram-positive pathogen Streptococcus pyogenes injects effector proteins into the cytosol of an infected host cell via the pore-forming protein streptolysin O is unknown. Key questions include whether the pathway can discriminate between different substrates for translocation, and whether the effector protein plays an active or passive role in the translocation process. Here we show that CMT can discriminate between a known effector of the pathway, the S. pyogenes NAD(+) glycohydrolase (SPN), and a second secreted protein, the mitogenic factor (MF), routing the former into the host cell cytosol and the latter into the extracellular milieu. Residues within the amino-terminal 190 residues of SPN were essential for discrimination, as deletions within this domain produced proteins that retained full enzymatic activity, but were completely uncoupled from the translocation pathway. The enzymatic domain itself played a pivotal role in the discrimination as deletions within this domain also produced translocation incompetent proteins and the conversion of MF to a translocation-competent form required fusion with both SPN domains in a contiguous orientation. These data establish that CMT is discriminatory, and that SPN is a multidomain protein that plays an active role in its translocation.     

Protective and inhibitory effects of various types of amphipols on the Ca2+-ATPase from sarcoplasmic reticulum: a comparative study

Amphipols are amphipathic polymers designed to replace or supplement detergents in membrane protein solution studies. Previous work has suggested both advantages and disadvantages to the use of a polyacrylate-based amphipol, A8-35, for studying the sarcoplasmic reticulum Ca2+-ATPase (SERCA1a). We investigated this issue further using a set of four amphipols with different chemical structures. Previous size exclusion chromatography experiments had shown that A8-35 and SERCA1a/A8-35 complexes aggregate under certain conditions. We show here that aggregation can be prevented by omitting calcium from buffers or by using a sulfonated version of A8-35. A8-35 had previously been shown to protect Ca2+-ATPase from irreversible denaturation, while inhibiting its activity in a reversible manner. We show here that the other three amphipols tested also display these properties and that all four amphipols slow down backward calcium dissociation from the nonphosphorylated solubilized enzyme, a priori an unrelated step. As this calcium dissociation involves the opening up of the bundle of transmembrane ATPase segments, the slowing of this process may indicate that multipoint attachment of the polymers to the hydrophobic transmembrane surface damps protein dynamics ("Gulliver" effect). Damping might be the reason why amphipols also simultaneously protect membrane proteins against irreversible denaturation and may inhibit the activity of those of them that display large rearrangements of their transmembrane surface during their catalytic cycle.     

Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions. The recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.     

Mitochondrial bovine ADP/ATP carrier in detergent is predominantly monomeric but also forms multimeric species

ADP/ATP carriers (AACs) are major and essential constituents of the inner mitochondrial membrane. They drive the import of ADP and the export of newly synthesized ATP. They were described as functional dimers from the 1980s until the structures of the AAC shed doubt on this consensus. We aimed to ascertain the published biophysical data claiming that AACs are dimers and to characterize the oligomeric state of the protein before crystallization. Analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine AAC is a monomer in 3-laurylamido-N,N'-dimethylpropylaminoxide (LAPAO), whereas in Triton X-100 and reduced Triton X-100, higher molecular mass species can also be identified. Neutron scattering data for monomeric bovine AAC in LAPAO does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. We discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of AACs within the mitochondrial membrane.     

A Locus for Autosomal Dominant Mitral Valve Prolapse on Chromosome 11p15.4

Mitral valve prolapse (MVP) is a common cardiovascular abnormality in the United States, occurring in approximately 2.4% of the general population. Clinically, patients with MVP exhibit fibromyxomatous changes in one or both of the mitral leaflets that result in superior displacement of the leaflets into the left atrium. Although often clinically benign, MVP can be associated with important accompanying sequelae, including mitral regurgitation, bacterial endocarditis, congestive heart failure, atrial fibrillation, and even sudden death. MVP is genetically heterogeneous and is inherited as an autosomal dominant trait that exhibits both sex- and age-dependent penetrance. In this report, we describe the results of a genome scan and show that a locus for MVP maps to chromosome 11p15.4. Multipoint parametric analysis performed by use of GENEHUNTER gave a maximum LOD score of 3.12 for the chromosomal region immediately surrounding the four-marker haplotype D11S4124-D11S2349-D11S1338-D11S1323, and multipoint nonparametric analysis (NPL) confirms this finding (NPL=38.59; P=.000397). Haplotype analysis across this region defines a 4.3-cM region between the markers D11S1923 and D11S1331 as the location of a new MVP locus, MMVP2, and confirms the genetic heterogeneity of this disorder. The discovery of genes involved in the pathogenesis of this common disease is crucial to understanding the marked variability in disease expression and mortality seen in MVP.     

In vivo developmental modifications of the expression of genes encoding muscle-specific enzymes in rat

cDNA clones for rat muscle-type creatine kinase and glycogen phosphorylase and aldolase A were isolated from a rat muscle cDNA library. An additional clone recognizing an unidentified 2.7-kilobase pair mRNA species was also isolated. These cDNA clones were used as probes to investigate the expression of the corresponding mRNAs during muscle development. Two aldolase A mRNA species were detected, one of 1650 bases expressed in non-muscle tissues, fetal muscle, and adult slow-twitch muscle, the other of 1550 bases was highly specific of adult fast-twitch skeletal muscle differentiation. These aldolase A mRNAs were shown by primer extension to differ by their 5' ends. The accumulation of muscle-type phosphorylase and creatine kinase and muscle-specific aldolase A mRNA accumulation during muscle development seems to be a coordinate process occurring progressively from the 17th day of intrauterine life up to the 30th day after birth. In contrast, the 2.7-kilobase pair RNA species is maximally expressed at the 1st week after birth as is the neonatal form of myosin heavy chain mRNA.     

Developmental regulation of the aldolase A muscle-specific promoter during in vivo muscle maturation is controlled by a nuclear receptor binding element.

During the post-natal period, skeletal muscles undergo important modifications leading to the appearance of different types of myofibers which exhibit distinct contractile and metabolic properties. This maturation process results from the activation of the expression of different sets of contractile proteins and metabolic enzymes, which are specific to the different types of myofibers. The muscle-specific promoter of the aldolase A gene (pM) is expressed mainly in fast-twitch glycolytic fibers in adult body muscles. We investigate here how pM is regulated during the post-natal development of different types of skeletal muscles (slow or fast-twitch muscles, head or body muscles). We show that pM is expressed preferentially in prospective fast-twitch muscles soon after birth; pM is up-regulated specifically in body muscles only later in development. This activation pattern is mimicked by a transgene which comprises only the 355 most proximal sequences of pM. Within this region, we identify a DNA element which is required for the up-regulation of the transgene during post-natal development in body muscles. Comparison of nuclear M1-binding proteins from young or adult body muscles show no qualitative differences. Distinct M1-binding proteins are present in both young and adult tongue nuclear extracts, compared to that present in gastrocnemius extracts.     

Transport mechanism of the sarcoplasmic reticulum Ca2+ -ATPase pump

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) belongs to the group of P-type ATPases, which actively transport inorganic cations across membranes at the expense of ATP hydrolysis. Three-dimensional structures of several transport intermediates of SERCA1a, stabilized by structural analogues of ATP and phosphoryl groups, are now available at atomic resolution. This has enabled the transport cycle of the protein to be described, including the coupling of Ca(2+) occlusion and phosphorylation by ATP, and of proton counter-transport and dephosphorylation. From these structures, Ca(2+)-ATPase gradually emerges as a molecular mechanical device in which some of the transmembrane segments perform Ca(2+) transport by piston-like movements and by the transmission of reciprocating movements that affect the chemical reactivity of the cytosolic globular domains.