Mammalian end binding proteins control persistent microtubule growth

© 2009 Komarova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0. The definitive version was published in Journal of Cell Biology 184 (2009): 691-706, doi:10.1083/jcb.200807179.End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.This work was supported by the Netherlands Organization for Scientifi c Research grants to A.A., by Funda ç ã o para a Ci ê ncia e a Tecnologia fellowship to S.M. Gouveia, by a FEBS fellowship to R.M. Buey, by the National Institutes of Health grant GM25062 to G.G. Borisy and by the Swiss National Science Foundation through grant 3100A0-109423 and by the National Center of Competence in Research Structural Biology program to M.O. Steinmetz

Mutations in sphingosine-1-phosphate lyase cause nephrosis with ichthyosis and adrenal insufficiency

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease cases. A mutation in 1 of over 40 monogenic genes can be detected in approximately 30% of individuals with SRNS whose symptoms manifest before 25 years of age. However, in many patients, the genetic etiology remains unknown. Here, we have performed whole exome sequencing to identify recessive causes of SRNS. In 7 families with SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency, and neurological defects, we identified 9 different recessive mutations in SGPL1, which encodes sphingosine-1-phosphate (S1P) lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing SGPL1 mutations resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1. yeast strains, whereas expression of disease-associated variants did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of Sgpl1 in rat mesangial cells inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. In Drosophila, Sply mutants, which lack SGPL1, displayed a phenotype reminiscent of nephrotic syndrome in nephrocytes. WT Sply, but not the disease-associated variants, rescued this phenotype. Together, these results indicate that SGPL1 mutations cause a syndromic form of SRNS

Manuscript: SGPL1 mutations cause nephrosis with ichthyosis and adrenal insufficiency. Note: Details of NIBR contribution to manuscript: The results of Mutational analysis of SGPL1: In silico modelling ( were approved in OAK before-ref link https://oak-intra.novartis.com/25460/). Mutational analysis of SGPL1: In silico modelling results were shared with Prof. Friedhelm Hildebrandt, Harvard medical school, Division of Nephrology Boston Children's Hospital. These results, that were shared with Prof. Friedhelm Hildebrandt is now a part of full manuscript for publication with Prof. Friedhelm Hildebrandt as corresponding author . Honnappa Srinivas and Rainer Wilcken are the co-authors from NIBR. Please find attached the full manuscript.

ABSTRACT Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease. A mutation in one of >40 different monogenic genes can be detected in ~30% of individuals with SRNS who manifest before 25 years of age. However, in many patients the genetic etiology remains unknown. We performed whole exome sequencing (WES) to identify novel recessive causes of SRNS. In 7 families with a new syndrome of SRNS and facultative ichthyosis, adrenal insufficiency, immunodeficiency and neurological defects, we identified 9 different recessive mutations in SGPL1 encoding sphingosine-1-phosphate lyase. All mutations resulted in reduced or absent SGPL1 protein and/or enzyme activity. Overexpression of cDNA representing mutations of SRNS patients resulted in subcellular mislocalization of SGPL1. Furthermore, expression of WT human SGPL1 rescued growth of SGPL1-deficient dpl1 yeast strains, whereas expression of disease-associated variant proteins did not. Immunofluorescence revealed SGPL1 expression in mouse podocytes and mesangial cells. Knockdown of SGPL1 in rat mesangial cells (RMC) inhibited cell migration, which was partially rescued by VPC23109, an S1P receptor antagonist. Knockdown of SGPL1 in RMC resulted in a decrease of active RAC1 and CDC42, consistent with previously noted podocytic imbalance of active RAC1 in the pathogenesis of SRNS. In Drosophila Sply mutants, which lack SGPL1, a phenotype reminiscent of nephrotic syndrome was observed in ‘nephrocytes’ and was rescued by WT Sply but not by the disease-associated variants. We here discover SGPL1 mutations as a new syndromic form of SRNS. Its pathogenesis entails mesangial cell dysfunction and decrease of active RAC1, which was partially mitigated by S1P receptor inhibition

Mutational analysis of SGPL1: In silico modelling (The analysis results will be shared with Prof. Friedhelm Hildebrandt, Harvard medical school, Division of Nephrology Boston Children's Hospital). These results ( were approved in OAK before), that were shared with Prof. Friedhelm Hildebrandt is now a part of full manuscript for publication with Prof. Friedhelm Hildebrandt as corresponding author . Honnappa Srinivas and Rainer Wilken are the co-authors from NIBR. Please find attached the full manuscript.

Sphingosine-1-phosphate lyase (SGPL1), a key enzyme of sphingolipid metabolism, catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. . Its main substrate sphingosine-1-phosphate (S1P) acts both extracellularly, by binding G protein-coupled receptors of the lysophospholipid receptor family, and inside the cell, as a second messenger. Therefore, S1P takes part in regulating various cellular processes and its levels are tightly regulated. SGPL1 is a symmetric homodimer; two subunits form a tightly intertwined dimer with both chains contributing to the catalytic cavity defined by the covalently bound cofactor pyridoxal phosphate (PLP). Two mutations (R222Q and S346Ile) were found in individuals from families with nephrotic syndrome (unpublished data, Prof. Friedhelm Hildebrandt, Harvard medical school, Division of Nephrology Boston Children's Hospital). To understand the structural changes due to these mutations, we have performed an in silico modelling analysis. The published SGPL1 structure (PDB accession 4Q6R) served as a basis for our in silico model analysis to derive qualitative measure of protein stability and dimer affinity

Interactions of substrate with calreticulin, an endoplasmic reticulum chaperone

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc1Man7-9GlcNAc2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc1Man7-9GlcNAc2, determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (Kb ) and changes in enthalpy of binding (ΔHb°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19×104 M−1 at 279 K is obtained when glucose occurs in α-1,3 linkage to ManαMe as in Glcα1-3ManαMe. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glcα1-3Manα1-2Manα1-2ManαMe structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis à vis legume lectins, which have the same fold as this chaperone, are discussed

Interactions of Substrate with Calreticulin, an Endoplasmic Reticulum Chaperone

Calreticulin is a molecular chaperone found in the endoplasmic reticulum in eukaryotes, and its interaction with N-glycosylated polypeptides is mediated by the glycan Glc1Man7-9GlcNAc2 present on the target glycoproteins. Here, we report the thermodynamic parameters of its interaction with di-, tri-, and tetrasaccharide, which are truncated versions of the glucosylated arm of Glc1Man7-9GlcNAc2, determined by the quantitative technique of isothermal titration calorimetry. This method provides a direct estimate of the binding constants (Kb) and changes in enthalpy of binding (Delta Hb°) as well as the stoichiometry of the reaction. Unlike past speculations, these studies demonstrate unambiguously that calreticulin has only one site per molecule for binding its complementary glucosylated ligands. Although the binding of glucose by itself is not detectable, a binding constant of 4.19 × 104 M-1 at 279 K is obtained when glucose occurs in alpha -1,3 linkage to Manalpha Me as in Glcalpha 1-3Manalpha Me. The binding constant increases by 25-fold from di- to trisaccharide and doubles from tri- to tetrasaccharide, demonstrating that the entire Glcalpha 1-3Manalpha 1-2Manalpha 1-2Manalpha Me structure of the oligosaccharide is recognized by calreticulin. The thermodynamic parameters thus obtained were supported by modeling studies, which showed that increased number of hydrogen bonds and van der Waals interactions occur as the size of the oligosaccharide is increased. Also, several novel findings about the recognition of saccharide ligands by calreticulin vis á vis legume lectins, which have the same fold as this chaperone, are discussed

A conserved trimerization motif controls the topology of short coiled coils

In recent years, short coiled coils have been used for applications ranging from biomaterial to medical sciences. For many of these applications knowledge of the factors that control the topology of the engineered protein systems is essential. Here, we demonstrate that trimerization of short coiled coils is determined by a distinct structural motif that encompasses specific networks of surface salt bridges and optimal hydrophobic packing interactions. The motif is conserved among intracellular, extracellular, viral, and synthetic proteins and defines a universal molecular determinant for trimer formation of short coiled coils. In addition to being of particular interest for the biotechnological production of candidate therapeutic proteins, these findings may be of interest for viral drug development strategies