Nucleotide-dependent shape changes in the reverse direction motor, myosin VI

We have studied the shape of myosin VI, the actin minus-end directed motor, by negative stain and metal shadow electron microscopy. Single particle processing was used to make two-dimensional averages of the stain images, which greatly increases the clarity and allows detailed comparisons with crystal structures. A total of 169,964 particle images were obtained from two different constructs in six different states (four nucleotide states and with and without Ca²⁺). The shape of truncated apo myosin VI was very similar to the apo crystal structure, with the lever arm bent strongly backward and around the motor domain. In the full-length molecule, the C-terminal part of the tail has an additional bend taking it back across the motor domain, which may reflect a regulated state. Addition of ATP, ADP, or ATP-γS resulted in a large change, straightening the molecule from the bent shape and swinging the lever by ∼140°. Although these nucleotides would not be expected to produce the pre-powerstroke state, myosin VI in their presence was most similar to the truncated crystal structure with bound ADP-VO₄, which is thought to show the pre-powerstroke shape. The nucleotide data were therefore substantially different from expectation based on crystal structures. The full-length molecule was almost completely monomeric; only ∼1% were dimers, joined through the ends of the tail. Addition of calcium ions appeared to result in release of the second calmodulin light chain. In negatively stained molecules there was little indication of extended α-helical structure in the tail, but molecules viewed by metal shadowing had a tail ∼3× longer, 29 vs. 9 nm, part of which is likely to be a single α-helix.     

Final height in patients with idiopathic short stature and high growth hormone responses to stimulation tests

Children with idiopathic short stature (ISS) may have normal or increased growth hormone (GH) responses to provocation tests and achieve a final height (FH) below -2.0 standard deviation score (SDS) if untreated. FH of subjects with high stimulated GH levels has not been studied in detail. AIM: It was the aim of this study to analyse FH in ISS patients with high GH peak responses to the provocation test. PATIENTS AND METHODS: We studied 16 patients (9 pre-pubertal) with ISS and a GH peak >or=40 mU/l to insulin-induced hypoglycaemia. The patients were recalled at age 19.7 +/- 2.5 years for measurement of FH when blood samples were obtained for serum insulin-like growth factor (IGF)-I, IGF binding protein 3, acid-labile subunit and GH binding protein measurements. GH bioactivity was determined using the Nb2 bioassay. RESULTS: FH was -3.1 +/- 1.0 SDS, being significantly lower than target height (TH). At FH, IGF-I levels were within -1.5 and +1.5 SDS for age and sex in 10 patients and higher than +1.5 SDS in 6 patients. IGF binding protein 3, acid-labile subunit, GH binding protein levels and GH bioactivity values were normal. SUMMARY: These data suggest that patients with ISS and high GH levels during a GH stimulation test may have a more compromised FH. The association of severe ISS with a peak GH >40 mU/l might suggest a degree of insensitivity for the GH-IGF-I axis.     

CRB2 Mutations Produce a Phenotype Resembling Congenital Nephrosis,Finnish Type,with Cerebral Ventriculomegaly and Raised Alpha-Fetoprotein

We report five fetuses and a child from three families who shared a phenotype comprising cerebral ventriculomegaly and echogenic kidneys with histopathological findings of congenital nephrosis. The presenting features were greatly elevated maternal serum alpha-fetoprotein (MSAFP) or amniotic fluid alpha-fetoprotein (AFAFP) levels or abnormalities visualized on ultrasound scan during the second trimester of pregnancy. Exome sequencing revealed deleterious sequence variants in Crumbs, Drosophila, Homolog of, 2 (CRB2) consistent with autosomal-recessive inheritance. Two fetuses with cerebral ventriculomegaly and renal microcysts were compound heterozygotes for p.Asn800Lys and p.Trp759Ter, one fetus with renal microcysts was a compound heterozygote for p.Glu643Ala and p.Asn800Lys, and one child with cerebral ventriculomegaly, periventricular heterotopias, echogenic kidneys, and renal failure was homozygous for p.Arg633Trp in CRB2. Examination of the kidneys in one fetus showed tubular cysts at the corticomedullary junction and diffuse effacement of the epithelial foot processes and microvillous transformation of the renal podocytes, findings that were similar to those reported in congenital nephrotic syndrome, Finnish type, that is caused by mutations in nephrin (NPHS1). Loss of function for crb2b and nphs1 in Danio rerio were previously shown to result in loss of the slit diaphragms of the podocytes, leading to the hypothesis that nephrosis develops from an inability to develop a functional glomerular barrier. We conclude that the phenotype associated with CRB2 mutations is pleiotropic and that the condition is an important consideration in the evaluation of high MSAFP/AFAFP where a renal cause is suspected.     

Signatures of miR-181a on the Renal Transcriptome and Blood Pressure

MicroRNA-181a binds to the 3′ untranslated region of messenger RNA (mRNA) for renin, a rate-limiting enzyme of the renin-angiotensin system. Our objective was to determine whether this molecular interaction translates into a clinically meaningful effect on blood pressure and whether circulating miR-181a is a measurable proxy of blood pressure. In 200 human kidneys from the TRANScriptome of renaL humAn TissuE (TRANSLATE) study, renal miR-181a was the sole negative predictor of renin mRNA and a strong correlate of circulating miR-181a. Elevated miR-181a levels correlated positively with systolic and diastolic blood pressure in TRANSLATE, and this association was independent of circulating renin. The association between serum miR-181a and systolic blood pressure was replicated in 199 subjects from the Genetic Regulation of Arterial Pressure of Humans In the Community (GRAPHIC) study. Renal immunohistochemistry and in situ hybridization showed that colocalization of miR-181a and renin was most prominent in collecting ducts where renin is not released into the systemic circulation. Analysis of 69 human kidneys characterized by RNA sequencing revealed that miR-181a was associated with downregulation of four mitochondrial pathways and upregulation of 41 signaling cascades of adaptive immunity and inflammation. We conclude that renal miR-181a has pleiotropic effects on pathways relevant to blood pressure regulation and that circulating levels of miR-181a are both a measurable proxy of renal miR-181a expression and a novel biochemical correlate of blood pressure.     

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through defined developmental transitions into a pure population of highly expandable OPCs in 10 d. These OPCs robustly differentiate into myelinating oligodendrocytes in vitro and in vivo. Our results demonstrate that mouse pluripotent stem cells provide a pure population of myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development and drug screening.     

Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.     

Epithelial proteomics in multiple organs and tissues: similarities and variations between cells, organs, and diseases

Epithelial cells play an important role in physiological and pathophysiological situations, with organ-, tissue-, type-, and function-specific patterns. Proteome analysis has been used to study epithelial-origin diseases and identify novel prognostic, diagnostic, and therapeutic markers. The present review compares the variation of sample preparation for epithelial proteomic analysis, search similarities, and differences of epithelial proteomics between different cells, locations, and diseases. We focus on specificity of proteomic markers for epithelial-involved diseases. Proteomic alterations in epithelial cell lines were mapped to understand protein patterns, differentiation, oncogenesis, and pathogenesis of epithelial-origin diseases. Changes of proteomic patterns depend on different epithelial cell lines, challenges, and preparation. Epithelial protein profiles associated with intracellular locations and protein function. Epithelial proteomics has been greatly developed to link clinical questions, e.g., disease severity, biomarkers for disease diagnosis, and drug targets. There is an exciting and attractive start to link epithelial proteomics with histology of clinical samples. From the present review, we can find that most of disease-associated investigation of epithelial proteomics has been focused on epithelial-origin cancer. There is a significant gap of epithelial proteomics between acute and chronic organ injury, inflammation, and multiple organ dysfunction. Epithelial proteomics will provide powerful information on the relationships between biological molecules and disease mechanisms. Epithelial proteomics strategies and approaches should become more global, multidimensional, and systemic.     

HER2/ErbB2-induced Breast Cancer Cell Migration and Invasion Require p120 Catenin Activation of Rac1 and Cdc42

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.     

Differential mapping of the amino acids mediating agonist and antagonist coordination with the human thromboxane A2 receptor protein

Despite the well documented involvement of thromboxane A(2) receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally designed antagonists available for clinical use. To a large extent, this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. On this basis, the purpose of the current study was to identify the specific amino acid residues in the TPR protein that regulate ligand coordination and binding. The sites selected for mutation reside within or in close proximity to a region we previously defined as a TPR ligand binding region (i.e. the C terminus of the second extracellular loop and the leading edge of the fifth transmembrane domain). Mutation of these residues caused varying effects on the TPR-ligand coordination process. Specifically, the D193A, D193Q, and D193R mutants lost SQ29,548 (antagonist) binding and exhibited a dramatically reduced calcium response, which could not be restored by elevated U46619 (agonist) doses. The F184Y mutant lost SQ29,548 binding and exhibited a reduced calcium response (which could be restored by elevated U46619); and the T186A and S191T mutants lost SQ29,548 binding and retained a normal U46619-induced calcium response. Furthermore, these last three mutants also revealed a divergence in the binding of two structurally different antagonists, SQ29,548 and BM13.505. Two separate mutants that exhibited SQ29,548 binding yielded either a normal (F196Y) or reduced (S201T) U46619 response. Finally, mutation of other residues directly adjacent to those described above (e.g. E190A and F200A) produced no detectable effects on either SQ29,548 binding or the U46619-induced response. In summary, these results identify key amino acids (in particular Asp(193)) involved in TPR ligand coordination. These findings also demonstrate that TPR-specific ligands interact with different residues in the ligand-binding pocket.