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Background: Patients with a history of intravenous drug abuse included in an official opioid substitution program represent an important subgroup of patients with chronic hepatitis C. The objective of this study was to assess the efficacy of and adherence to treatment with peginterferon and ribavirin in Austrian patients on stable opioid substitution therapy (OST). Methods: This prospective, multicenter, observational, non-interventional trial (clinicaltrials.gov identifier, NCT01416610) included treatment-naïve patients with chronic hepatitis C on OST. Treatment consisted of peginterferon alpha-2a (PEGASYS®, 180 µg/week) plus ribavirin (COPEGUS®, 1000/1200 mg/day in genotypes (GT) 1/4 and 800 mg/day in GT 2/3) for 24-72 weeks, according to GT and viral response. Results: The intention-to-treat (ITT) population comprised 88 patients. Mean duration of therapy was 6.0 ± 2.8 months. Treatment was discontinued earlier than planned in 34 out of 88 patients (39%), mainly because of poor adherence or side effects of treatment. At the end of treatment 65/88 patients (74%) were PCR negative. During follow-up, 5 patients relapsed. Only 44/88 patients (50%) could be evaluated 24 weeks after the end of treatment. Sustained virologic response 24 weeks after end of therapy (SVR24) was documented in 39/88 patients (44%). If only patients were considered who finished treatment as planned and for whom results at follow-up week 24 were available, the SVR24 rate was 89% (32/36). Conclusion: Despite favorable prognostic factors, such as young age and a high proportion of GT3, SVR rates were low in this cohort of patients receiving OST, the main reason being poor adherence; however, in those patients completing treatment, the SVR rate was high. [ABSTRACT FROM AUTHOR]

Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2's role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects. [ABSTRACT FROM AUTHOR]

Dictyostelium discoideum harbors a short (CRN12) and a long coronin (CRN7) composed of one and two beta-propellers, respectively. They are primarily present in the cell cortex and cells lacking CRN12 ( corA) or CRN7 ( corB) have defects in actin driven processes. We compared the characteristics of a mutant cell line ( corA /corB) lacking CRN12 and CRN7 with the single mutants focusing on cytokinesis, phagocytosis, chemotaxis and development. Cytokinesis, uptake of small particles, and developmental defects were not enhanced in the corA /corB strain as compared to the single mutants, whereas motility and phagocytosis of yeast particles were more severely impaired. It appears that although both proteins affect the same processes they do not act in a redundant manner. Rather, they often act antagonistically, which is in accordance with their proposed roles in the actin cytoskeleton where CRN12 acts in actin disassembly whereas CRN7 stabilizes actin filaments and protects them from disassembly. [ABSTRACT FROM AUTHOR]

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin. [ABSTRACT FROM AUTHOR]

Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments. [ABSTRACT FROM AUTHOR]

Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state 1H-15N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an α-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by β-strands. [ABSTRACT FROM AUTHOR]

Many F-actin crosslinking proteins consist of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. In this study, we used single-molecule force spectroscopy to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the six Ig domains unfolds at lower forces than do those of all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to contour length changes in the single-molecule unfolding pattern. These changes allowed us to map the stable core of ~60 amino acids that constitutes the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod. [ABSTRACT FROM AUTHOR]

The heteronuclear 15N-NOE experiment was extensively tested with respect to statistical and systematic experimental error. The dependence of signal intensity in the NOE experiment and in the reference experiment on the saturation and relaxation time was experimentally investigated. The statistics of the experimental values were accessed by numerous repetitions of identical set-ups. As a model system a protein of typical size for NMR studies was chosen, i.e., a 120 amino acid residues containing fragment of the F-actin binding gelation factor (ABP-120). The fragment exhibits fast dynamics that are accessible with the 15N-NOE experiment with various amplitudes. The results of this study show that commonly used parameters are only adequate for accurate measurement of motions with moderate amplitude. Highly flexible parts require longer delay times and thus more experimental time than commonly used. On the other hand, a qualitative or semi-quantitative assessment of a protein's mobility on fast times scales can be obtained from rapidly recorded experiments with unusual short delay times. The findings of this study are of equal importance for highly accurate measurement of the 15N-NOE as well as for quick identification of mobile or even unstructured residues/parts of a protein. [ABSTRACT FROM AUTHOR]

Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins. [ABSTRACT FROM AUTHOR]

Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor on resonance assignment of the N-terminal domain of the cyclase-associated protein from Dictyostelium discoideum.

Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actinfragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic­type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 Å resolution. The three-dimensional fold reveals a catalytic module of ∼160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin­fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity. [ABSTRACT FROM AUTHOR]

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains. [ABSTRACT FROM AUTHOR]