Myosin filament assembly requires a cluster of four positive residues located in the rod domain

Myosin has an intrinsic ability to organize into ordered thick filaments that mediate muscle contraction. Here, we use surface plasmon resonance and light scattering analysis to further characterize the molecular determinants that guide myosin filament assembly. Both assays identify a cluster of lysine and arginine residues as important for myosin polymerization in vitro. Moreover, in cardiomyocytes, replacement of these charged residues by alanine severely affects the incorporation of myosin into the distal ends of the sarcomere. Our findings show that a novel assembly element with a distinct charge profile is present at the C-terminus of sarcomeric myosins.

Structured summary of protein interactions

WT LMMbinds to WT LMM by surface plasmon resonance (View Interaction)WT LMMbinds to CT2 LMM by surface plasmon resonance (View Interaction)WT LMMbinds to Alanine mutant LMM by surface plasmon resonance (View Interaction)WT LMM and WT LMMbind by light scattering (View Interaction)Alanine mutant LMM and Alanine mutant LMMbind by light scattering (View Interaction)WT LMM and Alanine mutant LMMbind by light scattering (View Interaction)     

Effects of pathogenic proline mutations on myosin assembly

Laing distal myopathy (MPD1) is a genetically dominant myopathy characterized by early and selective weakness of the distal muscles. Mutations in the MYH7 gene encoding for the β-myosin heavy chain are the underlying genetic cause of MPD1. However, their pathogenic mechanisms are currently unknown. Here, we measure the biological effects of the R1500P and L1706P MPD1 mutations in different cellular systems. We show that, while the two mutations inhibit myosin self-assembly in non-muscle cells, they do not prevent incorporation of the mutant myosin into sarcomeres. Nevertheless, we find that the L1706P mutation affects proper antiparallel myosin association by accumulating in the bare zone of the sarcomere. Furthermore, bimolecular fluorescence complementation assay shows that the α-helix containing the R1500P mutation folds into homodimeric (mutant/mutant) and heterodimeric [mutant/wild type (WT)] myosin molecules that are competent for sarcomere incorporation. Both mutations also form aggregates consisting of cytoplasmic vacuoles surrounding paracrystalline arrays and amorphous rod-like inclusions that sequester WT myosin. Myosin aggregates were also detected in transgenic nematodes expressing the R1500P mutation. By showing that the two MPD1 mutations can have dominant effects on distinct components of the contractile apparatus, our data provide the first insights into the pathogenesis of the disease.     

Neuropilin-1 is upregulated in Sj?gren’s syndrome and contributes to pathological neovascularization

Neuropilin-1 (NRP1) is a transmembrane co-receptor for members of the vascular endothelial growth factor family. Recent studies revealed an important role of NRP1 in angiogenesis and progression of many diseases. The role of NRP1 in the development of Sj?gren’s syndrome (SS), one of the most common rheumatic diseases, has not yet been investigated. Molecular studies and protein expression techniques were performed to elucidate the gene and protein expression profile of NRP1 in human salivary gland epithelial cells (SGEC) from primary SS. We used human microarrays and transient transfection with a mutant form of the negative inhibitory κBα proteins (IκBαDN) to investigate whether selective inhibition of nuclear Factor-κB (NF-κB) improves NRP1-mediated pro-angiogenic factors release from SS SGEC. The selective NRP1 function inhibition with an antibody to human NRP1, was employed to evaluate the therapeutic potential of targeting NRP1. We demonstrate that NRP1 is expressed in SGEC of both human healthy biopsies and in SS samples, and increased NRP1 expression in SS SGEC is significantly associated with pro-angiogenic factors release. Neutralizing anti-NRP1 antibody decreased pro-angiogenic factor production from SS SGEC and blocking NF-κB activation could be a way to inhibit NRP1-mediated angiogenesis in Sj?gren’s syndrome.     

Synthesis of new 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted and antiplatelet/antiphlogistic activities evaluation

In pursuing our research on some 2,4-diamino-benzopyranopyrimidines and 2-amino-5,6-dihydrobenzo[h]quinazolines, previously reported as antiplatelet and analgesic/anti-inflammatory agents respectively, we designed and synthesized a new series of 5,6-dihydrobenzo[h]quinazoline 2,4-diamino substituted. The insertion of amino substituents at positions 2 and 4 of the benzoquinazoline scaffold resulted in compounds endowed with a potent ASA-like antiplatelet activity, combined with an anti-inflammatory activity comparable, in some cases, to that of indomethacin, used as a reference drug.     

Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae

Suspension cultured cells of a blast-resistant rice genotype (Oryza sativa L. cv. Gigante Vercelli) were treated with cell wall hydrolysates prepared from the fungal pathogen Magnaporthe oryzae. As a consequence, a complex pattern of phenylalanine ammonia lyase time course specific activity levels was evident. Ion-exchange chromatographic fractionation of crude extracts suggested that the early (6 h) and the late (48-72 h after elicitation) increase of activity relied upon the sequential induction of two different isoenzymes. The relative expression levels of 11 genes putatively coding for a phenylalanine ammonia lyase were measured by semi-quantitative capillary gel electrophoresis of RT-PCR products. Two genes were indeed found to be induced by treatments with the hydrolysate, and data were validated by real-time PCR. Conversely, only the early-responsive enzyme form was observed following elicitation in a blast-sensitive rice genotype (cv. Vialone nano). Therefore, the late-responsive isoform may represent a candidate gene to select for decreased sensitivity to blast.     

Ionizing radiations increase the activity of the cell surface glycohydrolases and the plasma membrane ceramide content

We detected significant levels of β-glucosidase, β-galactosidase, sialidase Neu3 and sphingomyelinase activities associated with the plasma membrane of fibroblasts from normal and Niemann-Pick subjects and of cells from breast, ovary, colon and neuroblastoma tumors in culture. All of the cells subjected to ionizing radiations showed an increase of the activity of plasma membrane β-glucosidase, β-galactosidase and sialidase Neu3, in addition of the well known increase of activity of plasma membrane sphingomyelinase, under similar conditions. Human breast cancer cell line T47D was studied in detail. In these cells the increase of activity of β-glucosidase and β-galactosidase was parallel to the increase of irradiation dose up to 60?Gy and continued with time, at least up to 72?h from irradiation. β-glucosidase increased up to 17 times and β-galactosidase up to 40 times with respect to control. Sialidase Neu3 and sphingomyelinase increased about 2 times at a dose of 20?Gy but no further significant differences were observed with increase of radiation dose and time. After irradiation, we observed a reduction of cell proliferation, an increase of apoptotic cell death and an increase of plasma membrane ceramide up to 3 times, with respect to control cells. Tritiated GM3 ganglioside has been administered to T47D cells under conditions that prevented the lysosomal catabolism. GM3 became component of the plasma membranes and was transformed into LacCer, GlcCer and ceramide. The quantity of ceramide produced in irradiated cells was about two times that of control cells.     

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.     

Does interspecific competition with a stronger competitor explain the rarity of an endangered snake on a Mediterranean island?

Interspecific competition has been demonstrated to be an important shaping force for snake communities worldwide, but relatively few studies have investigated its occurrence and extent with island assemblages of snakes. In Sardinia (Mediterranean Sea), two species of whip snakes (Colubridae) co-occur, one of them being abundant and widespread (Hierophis viridiflavus) and the other being localized and critically endangered (Hemorrhois hippocrepis). A previous hypothesis suggested that the latter species would be confined to suboptimal habitats by the former species, which is a stronger competitor for food. As a consequence, He. hippocrepis would not only be rare but would also be smaller in body size in Sardinia than in other regions of its range where Hi. viridiflavus does not occur. In 1999?C2010, we studied habitat selection, food habits, and body sizes of these two snakes in sympatric populations, applying a suite of statistical tools including null models and Monte Carlo simulations. We observed that dietary habits were different between species (compared to Hi. viridiflavus, He. hippocrepis more frequently preyed upon rodents and less frequently on lizards, and within rodent prey, more frequently upon Rattus and less frequently upon Mus), but not in a way compatible with competitively induced trophic niche partitioning. The two species were similar in terms of prey size and predator-size?Cprey-size relationships. They also differed in habitat selection (maquis was the preferred habitat for both, but Hi. viridiflavus was found significantly more often than He. hippocrepis in grasslands, cultivations, and artificial pinewoods) but again in a non-competitively directed way. In addition, there was no evidence for the hypothesis that He. hippocrepis was confined to suboptimal habitats. Body sizes were similar between species (with males being significantly larger than females), and Sardinian He. hippocrepis were not smaller than conspecifics inhabiting regions without Hi. viridiflavus. Overall, our study showed that the two species differed in some aspects of their ecology, but these differences could not be due to competitive interactions. The conservation implications of these results are also discussed.