Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.     

Motor protein Myo1c is a podocyte protein that facilitates the transport of slit diaphragm protein Neph1 to the podocyte membrane

The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.     

Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation

Accumulating evidence suggests that mitogen-activated protein kinase signaling pathways form modular signaling complexes. Because the mixed lineage kinase dual leucine zipper-bearing kinase (DLK) is a large modular protein, structure-function analysis was undertaken to examine the role of DLK domains in macromolecular complex formation. DLK mutants were used to demonstrate that a DLK leucine zipper-leucine zipper interaction is necessary for DLK dimerization and to show that DLK dimerization mediated by the leucine zipper domain is prerequisite for DLK activity and subsequent activation of stress-activated protein kinase (SAPK). Heterologous mixed lineage kinase family members can be co-immunoprecipitated. However, the DLK leucine zipper domain interacted specifically only with the DLK leucine zipper domain; in contrast, DLK NH(2)-terminal region was sufficient to co-immunoprecipitate leucine zipper kinase and DLK. DLK has been shown to associate with the putative scaffold protein JIP1. This association occurred through the DLK NH(2)-terminal region and occurred independently of DLK catalytic activity. Although the DLK NH(2)-terminal region associated directly with JIP-1, this region did not interact directly with either DLK or leucine zipper kinase. Therefore, DLK may interact with heterologous mixed lineage kinase proteins via intermediary proteins. The NH(2)-terminal region of overexpressed DLK was required for activation of SAPK. These results provide evidence that protein complex formation is required for signal transduction from DLK to SAPK.     

Mixed lineage kinase-dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components.

It has been proposed that JNK-interacting proteins (JIP) facilitate mixed lineage kinase-dependent signal transduction to JNK by aggregating the three components of a JNK module. A new model for the assembly and regulation of these modules is proposed based on several observations. First, artificially induced dimerization of dual leucine zipper-bearing kinase (DLK) confirmed that DLK dimerization is sufficient to induce DLK activation. Secondly, under basal conditions, DLK associated with JIP is held in a monomeric, unphosphorylated and catalytically inactive state. Thirdly, JNK recruitment to JIP coincided with significantly decreased affinity of JIP and DLK. JNK promoted the dimerization, phosphorylation and activation of JIP-associated DLK. Similarly, treatment of cells with okadaic acid inhibited DLK association with JIP and resulted in DLK dimerization in the presence of JIP. In summary, JIP maintains DLK in a monomeric, unphosphorylated, inactive state. Upon stimulation, JNK-JIP binding affinity increases while JIP-DLK interaction affinity is attenuated. Dissociation of DLK from JIP results in subsequent DLK dimerization, autophosphorylation and module activation. Evidence is provided that this model holds for other MLK-dependent JNK modules.     

Visual insight into how low pH alone can induce actin-severing ability in gelsolin under calcium-free conditions

Gelsolin is a key actin cytoskeleton-modulating protein primarily regulated by calcium and phosphoinositides. In addition, low pH has also been suggested to activate gelsolin in the absence of Ca²⁺ ions, although no structural insight on this pathway is available except for a reported decrement in its diffusion coefficient at low pH. We also observed ∼1.6-fold decrease in the molecular mobility of recombinant gelsolin when buffer pH was lowered from 9 to 5. Analysis of the small angle x-ray scattering data collected over the same pH range indicated that the radius of gyration and maximum linear dimension of gelsolin molecules increased from 30.3 to 34.1 Å and from 100 to 125 Å, respectively. Models generated for each dataset indicated that similar to the Ca²⁺-induced process, low pH also promotes unwinding of this six-domain protein but only partially. It appeared that pH is able to induce extension of the G1 domain from the rest of the five domains, whereas the Ca²⁺-sensitive latch between G2 and G6 domains remains closed. Interestingly, increasing the free Ca²⁺ level to merely ∼40 nm, the partially open pH 5 shape “sprung open” to a shape seen earlier for this protein at pH 8 and 1 mm free Ca²⁺. Also, pH alone could induce a shape where the g3-g4 linker of gelsolin was open when we truncated the C-tail latch from this protein. Our results provide insight into how under physiological conditions, a drop in pH can fully activate the F-actin-severing shape of gelsolin with micromolar levels of Ca²⁺ available.     

Mapping of the plasminogen binding site of streptokinase with short synthetic peptides.

Although several recent studies employing various truncated fragments of streptokinase (SK) have demonstrated that the high-affinity interactions of this protein with human plasminogen (HPG) to form activator complex (SK-HPG) are located in the central region of SK, the exact location and nature of such HPG interacting site(s) is still unclear. In order to locate the "core" HPG binding ability in SK, we focused on the primary structure of a tryptic fragment of SK derived from the central region (SK143-293) that could bind as well as activate HPG, albeit at reduced levels in comparison to the activity of the native, full-length protein. Because this fragment was refractory to further controlled proteolysis, we took recourse to a synthetic peptide approach wherein the HPG interacting properties of 16 overlapping 20-mer peptides derived from this region of SK were examined systematically. Only four peptides from this set, viz., SK234-253, SK254-273, SK274-293, and SK263-282, together representing the contiguous sequence SK234-293, displayed HPG binding ability. This was established by a specific HPG-binding ELISA as well as by dot blot assay using 125I-labeled HPG. These results showed that the minimal sequence with HPG binding function resided between residues 234 and 293. None of the synthetic SK peptides was found to activate HPG, either individually or in combination, but, in competition experiments where each of the peptides was added prior to complex formation between SK and HPG, three of the HPG binding peptides (SK234-253, SK254-273, and SK274-293) inhibited strongly the generation of a functional activator complex by SK and HPG. This indicated that residues 234-293 in SK participate directly in intermolecular contact formation with HPG during the formation of the 1:1 SK-HPG complex. Two of the three peptides (SK234-253 and SK274-293), apart from interfering in SK-HPG complex formation, also showed inhibition of the amidolytic activity of free HPN by increasing the K(m) by approximately fivefold. A similar increase in K(m) for amidolysis by HPN as a result of complexation with SK has been interpreted previously to arise from the steric hinderance at or near the active site due to the binding of SK in this region. Thus, our results suggest that SK234-253 and SK274-293 also, like SK, bound close to the active site of HPN, an event that was reflected in the observed alteration in its substrate accessibility. By contrast, whereas the intervening peptide (SK254-273) could not inhibit amidolysis by free HPN, it showed a marked inhibition of the activation of "substrate" PG (human or bovine plasminogen) by activator complex, indicating that this particular region is intimately involved in interaction of the SK-HPG activator complex with substrate plasminogen during the catalytic cycle. This finding provides a rational explanation for one of the most intriguing aspects of SK action, i.e., the ability of the SK-HPG complex to catalyze selectively the activation of substrate molecules of PG to PN, whereas free HPN alone cannot do so. Taken together, the results presented in this paper strongly support a model of SK action in which the segment 234-293 of SK, by virtue of the epitopes present in residues 234-253 and 274-293, binds close to the active center of HPN (or, a cryptic active site, in the case of HPG) during the intermolecular association of the two proteins to form the equimolar activator complex; the segment SK254-273 present in the center of the core region then imparts an ability to the activator complex to interact selectively with substrate PG molecules during each PG activation cycle.     

Solution structure analysis of cytoplasmic domain of podocyte protein Neph1 using small/wide angle x-ray scattering (SWAXS)

Neph1 is present in podocytes, where it plays a critical role in maintaining the filtration function of the glomerulus, in part through signaling events mediated by its cytoplasmic domain that are involved in actin cytoskeleton organization. To understand the function of this protein, a detailed knowledge of the structure of the Neph1 cytoplasmic domain (Neph1-CD) is required. In this study, the solution structure of this domain was determined by small/wide angle x-ray scattering (SWAXS). Analysis of Neph1-CD by SWAXS suggested that this protein adopts a global shape with a radius of gyration and a maximum linear dimension of 21.3 and 70 Å, respectively. These parameters and the data from circular dichroism experiments were used to construct a structural model of this protein. The His-ZO-1-PDZ1 (first PDZ domain of zonula occludens) domain that binds Neph1-CD was also analyzed by SWAXS, to confirm that it adopts a global structure similar to its crystal structure. We used the SWAXS intensity profile, the structural model of Neph1-CD, and the crystal structure of ZO-1-PDZ1 to construct a structural model of the Neph1-CD·ZO-1-PDZ1 complex. Mapping of the intermolecular interactions suggested that in addition to the C-terminal residues Thr-His-Val, residues Lys-761 and Tyr-762 in Neph1 are also critical for stabilizing the complex. Estimated intensity values from the SWAXS data and in vivo and in vitro pull-down experiments demonstrated loss of binding to ZO-1 when these residues were individually mutated to alanines. Our findings present a structural model that provides novel insights into the molecular structure and function of Neph1-CD.     

Functional study of mammalian Neph proteins in Drosophila melanogaster

Neph molecules are highly conserved immunoglobulin superfamily proteins (IgSF) which are essential for multiple morphogenetic processes, including glomerular development in mammals and neuronal as well as nephrocyte development in D. melanogaster. While D. melanogaster expresses two Neph-like proteins (Kirre and IrreC/Rst), three Neph proteins (Neph1-3) are expressed in the mammalian system. However, although these molecules are highly abundant, their molecular functions are still poorly understood. Here we report on a fly system in which we overexpress and replace endogenous Neph homologs with mammalian Neph1-3 proteins to identify functional Neph protein networks required for neuronal and nephrocyte development. Misexpression of Neph1, but neither Neph2 nor Neph3, phenocopies the overexpression of endogenous Neph molecules suggesting a functional diversity of mammalian Neph family proteins. Moreover, structure-function analysis identified a conserved and specific Neph1 protein motif that appears to be required for the functional replacement of Kirre. Hereby, we establish D. melanogaster as a genetic system to specifically model molecular Neph1 functions in vivo and identify a conserved amino acid motif linking Neph1 to Drosophila Kirre function.     

Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.