Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation.

NET-1 is a key chemotropic ligand that signals commissural axon migration and change in direction. NET-1 and its receptor UNC-5B switch axon growth cones from attraction to repulsion. The biophysical properties of the NET-1 + UNC-5B complex have been poorly characterized. Using multi-wavelength-AUC by adding a fluorophore to UNC-5B, we were able to separate the UNC-5B sedimentation from NET-1. Using both multi-wavelength- and single-wavelength AUC, we investigated NET-1 and UNC-5B hydrodynamic parameters and complex formation. The sedimentation velocity experiments show that NET-1 exists in a monomer-dimer equilibrium. A close study of the association shows that NET-1 forms a pH-sensitive dimer that interacts in an anti-parallel orientation. UNC-5B can form equimolar NET-1 + UNC-5B heterocomplexes with both monomeric and dimeric NET-1.

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1.

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins -1 and -4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.

Association of a Novel ACTA1 Mutation With a Dominant Progressive Scapuloperoneal Myopathy in an Extended Family.

IMPORTANCE: New genomic strategies can now be applied to identify a diagnosis in patients and families with previously undiagnosed rare genetic conditions. The large family evaluated in the present study was described in 1966 and now expands the phenotype of a known neuromuscular gene. OBJECTIVE: To determine the genetic cause of a slowly progressive, autosomal dominant, scapuloperoneal neuromuscular disorder by using linkage and exome sequencing. DESIGN, SETTING, AND PARTICIPANTS: Fourteen affected individuals in a 6-generation family with a progressive scapuloperoneal disorder were evaluated. Participants were examined at pediatric, neuromuscular, and research clinics from March 1, 2005, to May 31, 2014. Exome and linkage were performed in genetics laboratories of research institutions. MAIN OUTCOMES AND MEASURES: Examination and evaluation by magnetic resonance imaging, ultrasonography, electrodiagnostic studies, and muscle biopsies (n = 3). Genetic analysis included linkage analysis (n = 17) with exome sequencing (n = 7). RESULTS: Clinical findings included progressive muscle weakness in an initially scapuloperoneal and distal distribution, including wrist extensor weakness, finger and foot drop, scapular winging, mild facial weakness, Achilles tendon contractures, and diminished or absent deep tendon reflexes. Both age at onset and progression of the disease showed clinical variability within the family. Muscle biopsy specimens demonstrated type I fiber atrophy and trabeculated fibers without nemaline rods. Analysis of exome sequences within the linkage region (4.8 megabases) revealed missense mutation c.591C>A p.Glu197Asp in a highly conserved residue in exon 4 of ACTA1. The mutation cosegregated with disease in all tested individuals and was not present in unaffected individuals. CONCLUSIONS AND RELEVANCE: This family defines a new scapuloperoneal phenotype associated with an ACTA1 mutation. A highly conserved protein, ACTA1 is implicated in multiple muscle diseases, including nemaline myopathy, actin aggregate myopathy, fiber-type disproportion, and rod-core myopathy. To our knowledge, mutations in Glu197 have not been reported previously. This residue is highly conserved and located in an exposed position in the protein; the mutation affects the intermolecular and intramolecular electrostatic interactions as shown by structural modeling. The mutation in this residue does not appear to lead to rod formation or actin accumulation in vitro or in vivo, suggesting a different molecular mechanism from that of other ACTA1 diseases.

Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway.

Apoptins: selective anticancer agents.

Therapies that selectively target cancer cells for death have been the center of intense research recently. One potential therapy may involve apoptin proteins, which are able to induce apoptosis in cancer cells leaving normal cells unharmed. Apoptin was originally discovered in the Chicken anemia virus (CAV); however, human gyroviruses (HGyV) have recently been found that also harbor apoptin-like proteins. Although the cancer cell specific activity of these apoptins appears to be well conserved, the precise functions and mechanisms of action are yet to be fully elucidated. Strategies for both delivering apoptin to treat tumors and disseminating the protein inside the tumor body are now being developed, and have shown promise in preclinical animal studies.

Relaxin enhances the collagenolytic activity and in vitro invasiveness by upregulating matrix metalloproteinases in human thyroid carcinoma cells.

In this study, we identified differential expression of immunoreactive matrix metalloproteinase 2 (MMP2)/gelatinase A, membrane-anchored MT1-MMP/MMP14, and human relaxin-2 (RLN2) in human benign and malignant thyroid tissues. MMP2 and MT1-MMP were detected in the majority of thyroid cancer tissues and colocalized with RLN2-positive cells. MMP2 was mostly absent in goiter tissues and, similar to RLN2, may serve as a marker for thyroid cancer. MMP2 and MT1-MMP were identified as novel RLN2 targets. RLN2 caused a significant downregulation of tissue inhibitor of MMP (TIMP) 3 protein levels but did not change the expression levels of MMP13, and TIMP1, TIMP2, and TIMP4 in human thyroid carcinoma cells. RLN2 failed to affect the expression of MMP1, 3, 8, and 9 in the thyroid carcinoma cells investigated. Stable RLN2 transfectants secreted enhanced levels of bioactive MMP2 which contributed to the increased collagenolytic activity and in vitro invasiveness into collagen matrix by human thyroid cancer cells. Three-dimensional reconstitution of confocal fluorescent microscopy images revealed larger-sized invadopodia, with intense MT1-MMP accumulation at the leading migrating edge in RLN2 transfectants when compared with enhanced green fluorescent protein clones. In RLN2 transfectants actin stress fibers contributed to pseudopodia formation. In conclusion, enhanced tumor cell invasion by RLN2 involves the formation of MT1-MMP-enriched invadopodia that lead to increased collagenolytic cell invasion by human thyroid cancer cells.

Apoptin, a tumor-selective killer.

Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing insights for the development of novel tumor-selective anticancer drugs.