Kalirin Dbl-Homology Guanine Nucleotide Exchange Factor 1 Domain Initiates New Axon Outgrowths via RhoG-Mediated Mechanisms

The large multidomain Kalirin and Trio proteins containing dual Rho GTPase guanine nucleotide exchange factor (GEF) domains have been implicated in the regulation of neuronal fiber extension and pathfinding during evelopment. In mammals, Kalirin is expressed predominantly in the nervous system, whereas Trio, broadly expressed throughout the body, is expressed at a lower level in the nervous system. To evaluate the role of Kalirin in fiber initiation and outgrowth, we microinjected cultured sympathetic neurons with vectors encoding Kalirin or with Kalirin antisense oligonucleotides, and we assessed neuronal fiber growth in a serum-free, satellite cell-free environment. Kalirin antisense oligonucleotides blocked the continued extension of preexisting axons. Kalirin overexpression induced the prolific sprouting of new axonal fibers that grew at the normal rate; the activity of Kalirin was entirely dependent on the activity of the first GEF domain. KalGEF1-induced sprouting of new fibers from lamellipodial structures was accompanied by extensive actin cytoskeleton reorganization. The kalGEF1 phenotype was mimicked by constitutively active RhoG and was blocked by RhoG inhibitors. Constitutively active Rac1, RhoA, and Cdc42 were unable to initiate new axons, whereas dominant-negative Rac1, RhoA, and Cdc42 failed to block axon sprouting. Thus Kalirin, acting via RhoG in a novel manner, plays a central role in establishing the morphological phenotypic diversity that is essential to the connectivity of the developing nervous system

Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): Structural and computational study

Many bioactive peptides, such as hormones and neuropeptides, require amidation at the C terminus for their full biological activity. Peptidylglycine a-hydroxylating monooxygenase (PHM) performs the first step of the amidation reaction-the hydroxylation of peptidylglycine substrates at the Ca position of the terminal glycine. The hydroxylation reaction is copper- and O2-dependent and requires 2 equiv of exogenous reductant. The proposed mechanism suggests thatO2 is reduced by two electrons, each provided by one of two nonequivalent copper sites in PHM (CuH and CuM). The characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate remain uncertain. To further investigate the nature of the key intermediates in the PHM cycle, we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.98-A° -resolution structure (hydro)peroxide binds solely to CuM in a slightly asymmetric side-on mode. The O-O interatomic distance of the copperbound ligand is 1.5 A ° , characteristic of peroxide/hydroperoxide species, and the Cu-O distances are 2.0 and 2.1 A ° . Density functional theory calculations using the first coordination sphere of the CuM active site as a model system show that the computed energies of the side-on L3CuM(II)-O2 2- species and its isomeric, end-on structure L3CuM(I)-O2 - are similar, suggesting that both these intermediates are significantly populated within the protein environment. This observation has important mechanistic implications. The geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O2 2- is in good agreement with the results of a hybrid quantum mechanical-molecular mechanical optimization of this species.Fil: Rudzka, Katarzyna. University Johns Hopkins; Estados UnidosFil: Moreno, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentina. Universidad de Buenos Aires; ArgentinaFil: Eipper, Betty. University Of Connecticut; Estados UnidosFil: Mains, Richard. University Of Connecticut; Estados UnidosFil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires; ArgentinaFil: Amzel, L. Mario. University Johns Hopkins; Estados Unido

Microvillar and ciliary defects in zebrafish lacking an actin-binding bioactive peptide amidating enzyme

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 8 (2018): 4547, doi:10.1038/s41598-018-22732-9.The assembly of membranous extensions such as microvilli and cilia in polarized cells is a tightly regulated, yet poorly understood, process. Peptidylglycine α-amidating monooxygenase (PAM), a membrane enzyme essential for the synthesis of amidated bioactive peptides, was recently identified in motile and non-motile (primary) cilia and has an essential role in ciliogenesis in Chlamydomonas, Schmidtea and mouse. In mammalian cells, changes in PAM levels alter secretion and organization of the actin cytoskeleton. Here we show that lack of Pam in zebrafish recapitulates the lethal edematous phenotype observed in Pam−/− mice and reveals additional defects. The pam−/− zebrafish embryos display an initial striking loss of microvilli and subsequently impaired ciliogenesis in the pronephros. In multiciliated mouse tracheal epithelial cells, vesicular PAM staining colocalizes with apical actin, below the microvilli. In PAM-deficient Chlamydomonas, the actin cytoskeleton is dramatically reorganized, and expression of an actin paralogue is upregulated. Biochemical assays reveal that the cytosolic PAM C-terminal domain interacts directly with filamentous actin but does not alter the rate of actin polymerization or disassembly. Our results point to a critical role for PAM in organizing the actin cytoskeleton during development, which could in turn impact both microvillus formation and ciliogenesis.This study was supported by grants DK032949 (to BAE and REM), DK044464 (to JDG) and GM051293 (to SMK) from the National Institutes of Health

The Rho-GEF Kalirin regulates bone mass and the function of osteoblasts and osteoclasts

Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts. Dysregulation in the activity of the bone cells can lead to osteoporosis, a disease characterized by low bone mass and an increase in bone fragility and risk of fracture. Kalirin is a novel GTP-exchange factor protein that has been shown to play a role in cytoskeletal remodeling and dendritic spine formation in neurons. We examined Kalirin expression in skeletal tissue and found that it was expressed in osteoclasts and osteoblasts. Furthermore, micro-CT analyses of the distal femur of global Kalirin knockout (Kal-KO) mice revealed significantly reduced trabecular and cortical bone parameters in Kal-KO mice, compared to WT mice, with significantly reduced bone mass in 8, 14 and 36 week-old female Kal-KO mice. Male mice also exhibited a decrease in bone parameters but not to the level seen in female mice. Histomorphometric analyses also revealed decreased bone formation rate in 14 week-old female Kal-KO mice, as well as decreased osteoblast number/bone surface and increased osteoclast surface/bone surface. Consistent with our in vivo findings, the bone resorbing activity and differentiation of Kal-KO osteoclasts was increased in vitro. Although alkaline phosphatase activity by Kal-KO osteoblasts was increased in vitro, Kal-KO osteoblasts showed decreased mineralizing activity, as well as decreased secretion of OPG, which was inversely correlated with ERK activity. Taken together, our findings suggest that deletion of Kalirin directly affects osteoclast and osteoblast activity, leading to decreased OPG secretion by osteoblasts which is likely to alter the RANKL/OPG ratio and promote osteoclastogenesis. Therefore, Kalirin may play a role in paracrine and/or endocrine signaling events that control skeletal bone remodeling and the maintenance of bone mass

Captivated

Captivate

Retroflection

Retroflectio

Screenspeak: The Revenge of the Oral

Screenspeak: The Revenge of the Ora

A novel auxiliary subunit critical to BK channel function in caenorhabditis elegans

The BK channel is a Ca(2+)- and voltage-gated potassium channel with many important physiological functions. To identify proteins important to its function in vivo, we screened for C. elegans mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of the BK channel α-subunit SLO-1. BKIP-1, a small peptide with no significant homology to any previously characterized molecules was thus identified. BKIP-1 and SLO-1 showed similar expression and subcellular localization patterns, and appeared to interact physically through discrete domains. bkip-1 loss-of-function (lf) mutants phenocopied slo-1(lf) mutants in behavior and synaptic transmission, and suppressed the lethargy, egg-laying defect, and deficient neurotransmitter release caused by SLO-1(gf). In heterologous expression systems, BKIP-1 decreased the activation rate and shifted the conductance-voltage (G-V) relationship of SLO-1 in a Ca(2+)-dependent manner, and increased SLO-1 surface expression. Thus, BKIP-1 is a novel auxiliary subunit critical to SLO-1 function in vivo