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Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson’s disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17 kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17 kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (P = .0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.

The QRS interval, from the beginning of the Q wave to the end of the S wave on an electrocardiogram, reflects ventricular depolarization and conduction time and is a risk factor for mortality, sudden death and heart failure. We performed a genomewide association meta-analysis in 40,407 individuals of European descent from 14 studies, with further genotyping in 7,170 additional Europeans, and we identified 22 loci associated with QRS duration ( P < 5 x 10(-8)). These loci map in or near genes in pathways with established roles in ventricular conduction such as sodium channels, transcription factors and calcium-handling proteins, but also point to previously unidentified biologic processes, such as kinase inhibitors and genes related to tumorigenesis. We demonstrate that SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. These findings extend our current knowledge of ventricular depolarization and conduction.

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.

Vinculin is found in all adherens junctions, while metavinculin, a larger splice variant, is coexpressed with vinculin only in smooth and cardiac muscle. To understand the significance of metavinculin expression, we compared ligand binding between turkey vinculin and metavinculin. Residues 1–258 were found essential for head-tail interactions in both proteins. The tail domains (VT and MVT, respectively) both bind to F-actin. However, while VT bundles F-actin, MVT generates highly viscous F-actin webs. In transfected PtK2 cells, VT causes F-actin needles or coils, while MVT-expressing cells display a diffuse F-actin distribution. Thus, the MVT-specific insert induces an F-actin supraorganization different from the VT-based form, suggesting that metavinculin has a specific role in muscle.

Twenty-one expressed sequence tags (ESTs) belonging to unidentified transcripts were mapped to 9 intervals at 11p 15.5-p15.3. Thirteen were mapped to a region of an estimated size of 7 Mb with the help of a YAC contig. The remaining eight were mapped outside this region, and the centromeric or telomeric position of the ESTs relative to the YAC contig was defined with the help of a cellular hybrid containing a derivative chromosome 11 with a break-point within the region covered by the contig. The average size of the intervals, where the ESTs were mapped, was estimated to be 0.3-0.4 Mb. The refinement of the chromosomal location of these ESTs could be of great help in the identification of potential candidate genes for disease locus mapping at 11p15.

Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vinculin is primarily phosphorylated within the tail domain and suggest a function of vinculin phosphorylation in the regulation of the vinculin conformation.

The osmotic behavior of myosin subfragment 1 was studied at 22 degrees C and pH 7.45 in 0.1 m KCl, 2 mm MgCl2, and 10 mm triethanolamine or in 25 mm phosphate, 2 mm MgCl2, and 2 mm MgADP. It was found that, in 0.1 m KCl, myosin subfragment 1 behaved as a spheroidal particle, with an average diameter of 8.09 nm, composed of two myosin subfragment 1 molecules. The lower limit of the thermodynamic dimerization constant was estimated to be 3.5 x 10(4) M-1. Above 5 mm as monomer, myosin subfragment 1 departed from the behavior expected of a dimeric spheroidal model because of the onset of a "hydration force." This force measured at the contact distance between particles equals 2.18 x 10(7) dynes/cm2 and falls off exponentially with a decay distance of 0.27 nm. In 25 mm orthophosphate, myosin subfragment 1, with an increase in the protein osmotic pressure, shifted from the behavior of a sphere to that of a cylinder. Between 1 x 10(5) and 4 x 10(5) dynes/cm2, the behavior of myosin subfragment 1 was different in the presence and in the absence of MgADP. In particular, at 1.8 x 10(5) dynes/cm2, the protein osmotic pressure in frog muscle, myosin subfragment 1 behaved as a sphere of 3.21-nm radius in the presence of MgADP and as a cylinder with a length to diameter ratio of 2.07 in the absence of MgADP. Under the solution conditions used in this work, S1 never behaved as a fully extended particle.

At the physiological protein osmotic pressure, the angle formed between the long axis of the actin monomer and the pointed end of the filament axis is roughly 61 degrees in F-actin and about 90 degrees in the myosin subfragment 1--decorated F-actin. This implies that, in the course of the contractile cycle, actin itself contributes, by about 4 nm, to the displacement of the actin filament toward the center of the sarcomer.

The diameter of the actin filament is influenced by osmotic stress, being 9.0 nm at 1×10 5 dynes/cm 2 and 6.8 nm at 9.00×10 6 dynes/cm 2 . At 1.81×10 5 dynes/cm 2 , the protein osmotic pressure in frog muscle, the diameter is 7.95 nm. The diameter of the tropomyosin-decorated actin filament is also influenced by osmotic stress even though, at the low pressures (up to 2×10 5 dynes/cm 2 ), the decorated filament is significantly more resistant to compression than the undecorated actin filament.

We have modeled the effect of protein osmotic pressure on the orientation of the monomer in F-actin, in tropomyosin-F-actin, in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin. According to the model, at the physiological protein osmotic pressure (18 kPa), the elastic moduli by bending of the monomer in F-actin and in tropomyosin-F-actin are calculated to be 4.74 MPa and 5.8 MPa, respectively. The elastic moduli by bending of the monomer in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin are calculated to be 22MPa and 22.3MPa, respectively. These latter values are in excellent agreement with the values of the elastic moduli by stretching found for the fibres of frog and rabbit muscle. We have also calculated that, at the physiological protein osmotic pressure, the myosin subfragment-1 decorated F-actin rigor complex can develop a force of 3.96 pN, a force correctly oriented to promote the sliding of the actin filament toward the center of the sarcomere. The magnitude of this force is comparable to that reported for intact skeletal muscle. In contrast, the myosin subfragment-1 decorated tropomyosin-F-actin rigor complex develops a much smaller driving force, that favours relaxation. Apparently tropomyosin uncouples the osmotic and the mechanical event. It is proposed that the elastic energy for muscle contraction is provided by protein osmotic pressure.

The effect of alpha-actinin from chicken gizzard on the properties of F-actin solutions at 37 degrees C and at 4 degrees C was investigated. Beside the well-known increase of the gelling activity of alpha-actinin, it was found that lowering temperature to 4 degrees C: (a) modifies the shape of the alpha-actinin-F-actin binding isotherm; (b) increases the light scattering of the alpha-actinin-F-actin mixtures; (c) induces the formation of ribbons and bundles of F-actin. It was also observed that, by warming to 37 degrees C, the bundles of F-actin formed at 4 degrees C were dissociated into quasi-parallel actin filaments running at a distance of 25-42 nm from each other. On subsequent cooling to 4 degrees C, these parallel filaments were rapidly assembled into bundles. As at 37 degrees C, alpha-actinin displays a potent gelling activity on bundles but not on filaments of actin, and as the gelling activity at 4 degrees C is accompanied by the formation of actin bundles, it is concluded that actin bundling is a necessary condition to promote the actin-gelling activity.

The interaction of alpha-actinin from chicken gizzard with F-actin is quite complex. The apparent dissociation constant, C, increases with the increase of actin concentration according to the following expression: C = Ko + a[actin] - c[actin]5/2. At pH 7.5 and 37 degrees C, in the presence of 0.1 M KCl and 2 mM MgCl2, the dissociation constant at infinite actin dilution, Ko, is 2.17 microM. The binding of alpha-actinin to actin is related by the term a[actin] to the diffusion of actin filaments and by the term c[actin]5/2 to the crossing number concentration of the F-actin network. Especially at low actin concentration, the binding of alpha-actinin to actin is increased by gelsolin, which fragments actin filaments and increases their diffusion. The different binding isotherms of alpha-actinin to actin filaments and to actin bundles are discussed.

Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p −8 ) for serum albumin ( HPN-SCN1B , GCKR-FNDC4 , SERPINF2-WDR81 , TNFRSF11A-ZCCHC2 , FRMD5-WDR76 , and RPS11-FCGRT , in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein ( TNFRS13B , 6q21.3, and ELL2 , in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN , for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease.