Deafness DFNB128 Associated with a Recessive Variant of Human Recapitulates Hearing Loss of -Deficient Mice.

Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many mutant genes causing rare forms of deafness remain to be discovered. A consanguineous Pakistani family segregating nonsyndromic deafness in two sibships were studied using microarrays and exome sequencing.

The kinesin-5 tail and bipolar minifilament domains are the origin of its microtubule crosslinking and sliding activity.

Kinesin-5 crosslinks and slides apart microtubules to assemble, elongate, and maintain the mitotic spindle. Kinesin-5 is a tetramer, where two N-terminal motor domains are positioned at each end of the motor, and the coiled-coil stalk domains are organized into a tetrameric bundle through the bipolar assembly (BASS) domain. To dissect the function of the individual structural elements of the motor, we constructed a minimal kinesin-5 tetramer (mini-tetramer).

Variants of LRP2, encoding a multifunctional cell-surface endocytic receptor, associated with hearing loss and retinal dystrophy.

Hereditary deafness and retinal dystrophy are each genetically heterogenous and clinically variable. Three small unrelated families segregating the combination of deafness and retinal dystrophy were studied by exome sequencing (ES). The proband of Family 1 was found to be compound heterozygous for NM_004525.

Unbalanced bidirectional radial stiffness gradients within the organ of Corti promoted by TRIOBP.

Hearing depends on intricate morphologies and mechanical properties of diverse inner ear cell types. The individual contributions of various inner ear cell types into mechanical properties of the organ of Corti and the mechanisms of their integration are yet largely unknown. Using sub-100-nm spatial resolution atomic force microscopy (AFM), we mapped the Young's modulus (stiffness) of the apical surface of the different cells of the freshly dissected P5-P6 cochlear epithelium from wild-type and mice lacking either Trio and F-actin binding protein (TRIOBP) isoforms 4 and 5 or isoform 5 only.

Flexible microtubule anchoring modulates the bi-directional motility of the kinesin-5 Cin8.

Two modes of motility have been reported for bi-directional kinesin-5 motors: (a) context-dependent directionality reversal, a mode in which motors undergo persistent minus-end directed motility at the single-molecule level and switch to plus-end directed motility in different assays or under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. Here, we examine how modulation of motor-microtubule (MT) interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop 8 (L8) within the motor domain of Cin8 increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions.

The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding.

G Protein-Coupled Receptor Kinase 2 (GRK2) and 5 (GRK5) Exhibit Selective Phosphorylation of the Neurotensin Receptor in Vitro.

G protein-coupled receptor kinases (GRKs) play an important role in the desensitization of G protein-mediated signaling of G protein-coupled receptors (GPCRs). The level of interest in mapping their phosphorylation sites has increased because recent studies suggest that the differential pattern of receptor phosphorylation has distinct biological consequences. In vitro phosphorylation experiments using well-controlled systems are useful for deciphering the complexity of these physiological reactions and understanding the targeted event.

Biophysical characterization of membrane proteins in nanodiscs.

Nanodiscs are self-assembled discoidal phospholipid bilayers surrounded and stabilized by membrane scaffold proteins (MSPs), that have become a powerful and promising tool for the study of membrane proteins. Even though their reconstitution is highly regulated by the type of MSP and phospholipid input, a biophysical characterization leading to the determination of the stoichiometry of MSP, lipid and membrane protein is essential. This is important for biological studies, as the oligomeric state of membrane proteins often correlates with their functional activity.

Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid.

Membrane lipids have been implicated to influence the activity of G-protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus, insight gained from rhodopsin signaling may not be simply translated to other nonvisual GPCRs.

The binding mode of ATP revealed by the solution structure of the N-domain of human ATP7A.

We report the solution NMR structures of the N-domain of the Menkes protein (ATP7A) in the ATP-free and ATP-bound forms. The structures consist of a twisted antiparallel six-stranded beta-sheet flanked by two pairs of alpha-helices. A protein loop of 50 amino acids located between beta 3 and beta 4 is disordered and mobile on the subnanosecond time scale.

Crystal structure of CO-sensing transcription activator CooA bound to exogenous ligand imidazole.

CooA is a CO-dependent transcriptional activator and transmits a CO-sensing signal to a DNA promoter that controls the expression of the genes responsible for CO metabolism. CooA contains a b-type heme as the active site for sensing CO. CO binding to the heme induces a conformational change that switches CooA from an inactive to an active DNA-binding form.

Crystallization and preliminary X-ray analysis of CooA from Carboxydothermus hydrogenoformans.

CooA, a homodimeric haem-containing protein, is responsible for transcriptional regulation in response to carbon monoxide (CO). It has a b-type haem as a CO sensor. Upon binding CO to the haem, CooA binds promoter DNA and activates expression of genes for CO metabolism.

Evidence for displacements of the C-helix by CO ligation and DNA binding to CooA revealed by UV resonance Raman spectroscopy.

The UV and visible resonance Raman spectra are reported for CooA from Rhodospirillum rubrum, which is a transcriptional regulator activated by growth in a CO atmosphere. CO binding to heme in its sensor domain causes rearrangement of its DNA-binding domain, allowing binding of DNA with a specific sequence. The sensor and DNA-binding domains are linked by a hinge region that follows a long C-helix.

Spectroscopic and redox properties of a CooA homologue from Carboxydothermus hydrogenoformans.

CooA is a CO-sensing transcriptional activator that contains a b-type heme as the active site for sensing its physiological effector, CO. In this study, the spectroscopic and redox properties of a new CooA homologue from Carboxydothermus hydrogenoformans (Ch-CooA) were studied. Spectroscopic and mutagenesis studies revealed that His-82 and the N-terminal alpha-amino group were the axial ligands of the Fe(III) and Fe(II) hemes in Ch-CooA and that the N-terminal alpha-amino group was replaced by CO upon CO binding.

A Novel Hemiporphyrazine Comprising Three Isoindolediimine and Three Thiadiazole Units.

Incorporation of three metal ions (Ni or Cu) in the macrocyclic ring and the formation of hexamers following a 3+3 approach are novel features of the hemiporphyrazines (one example shown) formed by the condensation of 2,5-diamino-1,3,4-triazole with isoindolediimines. This is in contrast to the corresponding reactions with diaminotriazoles, which afford 2+2 products.