Kiyotaka Nishikawa

Determination of the Specific Substrate Sequence Motifs of Protein Kinase C Isozymes

Protein kinase C (PKC) family members play significant roles in a variety of intracellular signal transduction processes, but information about the substrate specificities of each PKC family member is quite limited. In this study, we have determined the optimal peptide substrate sequence for each of nine human PKC isozymes (α, βI, βII, γ, δ, ε, η, μ, and ζ) by using an oriented peptide library. All PKC isozymes preferentially phosphorylated peptides with hydrophobic amino acids at position +1 carboxyl-terminal of the phosphorylated Ser and basic residues at position −3. All isozymes, except PKCμ, selected peptides with basic amino acids at positions −6, −4, and −2. PKCα, -βI, -βII, -γ, and -η selected peptides with basic amino acid at positions +2, +3, and +4, but PKCδ, -ε, -ζ, and -μ preferred peptides with hydrophobic amino acid at these positions. At position −5, the selectivity was quite different among the various isozymes; PKCα, -γ, and -δ selected peptides with Arg at this position while other PKC isozymes selected hydrophobic amino acids such as Phe, Leu, or Val. Interestingly, PKCμ showed extreme selectivity for peptides with Leu at this position. The predicted optimal sequences from position −3 to +2 for PKCα, -βI, -βII, -γ, -δ, and -η were very similar to the endogenous pseudosubstrate sequences of these PKC isozymes, indicating that these core regions may be important to the binding of corresponding substrate peptides. Synthetic peptides based on the predicted optimal sequences for PKCα, -βI, -δ, -ζ, and -μ were prepared and used for the determination of Km and Vmax for these isozymes. As judged by Vmax/Km values, these peptides were in general better substrates of the corresponding isozymes than those of the other PKC isozymes, supporting the idea that individual PKC isozymes have distinct optimal substrates. The structural basis for the selectivity of PKC isozymes is discussed based on residues predicted to form the catalytic cleft.

A therapeutic agent with oriented carbohydrates for treatment of infections by Shiga toxin-producing Escherichia coli O157:H7

Infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7, which causes diarrhea and hemorrhagic colitis in humans, often results in fatal systemic complications, such as neurological damage and hemolytic–uremic syndrome. Because Stx circulating in the blood is a major causative factor of these complications, the development of a Stx neutralizer that functions in the circulation holds promise as a viable therapy. Here we developed a series of carbosilane dendrimers, in which trisaccharides of globotriaosyl ceramide, a receptor for Stx, were variously oriented at their termini (referred to as SUPER TWIG), and identified a SUPER TWIG with six trisaccharides as a Stx neutralizer functioning in the circulation. This SUPER TWIG specifically bound to Stx with high affinity (Kd = 1.1 × 10−6 M) and inhibited the incorporation of the toxin into target cells. Intravenous administration of the SUPER TWIG along with Stx to mice substantially reduced the fatal brain damage and completely suppressed the lethal effect of Stx. Moreover, the SUPER TWIG protected mice from challenge with a fatal dose of E. coli O157:H7, even when administered after the establishment of the infection. The SUPER TWIG neutralized Stx in vivo by a mechanism in which the accumulation and immediate degradation of Stx by phagocytic macrophages present in the reticuloendothelial system were induced. Taken together, our findings indicate that this SUPER TWIG is therapeutic agent against infections by Stx-producing E. coli.

Oral Therapeutic Agents with Highly Clustered Globotriose for Treatment of Shiga Toxigenic Escherichia coli Infections

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

Protein kinase Cμ (PKCμ), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952–960), and its mechanism of regulation is not yet clear. Here we show that PKCμ forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCμ between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCμ failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCμ-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCμ including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCμ acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

Intracellular phosphatidylserine is essential for retrograde membrane traffic through endosomes

Phosphatidylserine (PS) is a relatively minor constituent of biological membranes. Despite its low abundance, PS in the plasma membrane (PM) plays key roles in various phenomena such as the coagulation cascade, clearance of apoptotic cells, and recruitment of signaling molecules. PS also localizes in endocytic organelles, but how this relates to its cellular functions remains unknown. Here we report that PS is essential for retrograde membrane traffic at recycling endosomes (REs). PS was most concentrated in REs among intracellular organelles, and evectin-2 (evt-2), a protein of previously unknown function, was targeted to REs by the binding of its pleckstrin homology (PH) domain to PS. X-ray analysis supported the specificity of the binding of PS to the PH domain. Depletion of evt-2 or masking of intracellular PS suppressed membrane traffic from REs to the Golgi. These findings uncover the molecular basis that controls the RE-to-Golgi transport and identify a unique PH domain that specifically recognizes PS but not polyphosphoinositides.

Role of mast cells in the development of renal fibrosis: Use of mast cell–deficient rats

Summers et al1 recently published their observations that mast cells are crucial to renal fibrosis induced by ureteral obstruction. This work confirmed our report showing that mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction (UUO) model2. Our manuscript was published online September 28th, 2011. We would like to take this opportunity to expand on this role of mast cells in renal fibrosis, and advance a hypothesis for the underlying mechanism. In view of the fact that mast cells are found in close proximity to fibroblasts in UUO kidneys, we hypothesize that mast cell mediators released during degranulation are responsible for fibroblast proliferation and activation ultimately leading to fibrosis3. Recent findings from our lab demonstrate that kidney fibroblasts express the ANG II AT1 receptor (R) and the histamine H1R subtypes. Addition of ANG II and histamine to kidney fibroblasts in culture, promote proliferation, TGF-βsynthesis and collagen production3. These results, along with the findings of Veerappan et al2 and Summers et al1 suggest that release of mast cell mediators like renin (ANG II) and histamine, provide a mechanism that couples mast cell degranulation to fibroblast activation with the ensuing fibrosis. Targeting of mast cells and their products may represent novel therapeutic targets for preventing renal fibrosis.

A multivalent peptide library approach identifies a novel Shiga toxin inhibitor that induces aberrant cellular transport of the toxin

Infection with Shiga toxin (Stx)‐producing Escherichia coli O157:H7 causes bloody diarrhea and hemorrhagic colitis in humans, sometimes resulting in fatal systemic complications. Among the known Stx family members, Stx2 is responsible for the most severe forms of disease. Stx2 binds to target cells via multivalent interactions between its B‐subunit pentamer and globotriaosyl ceramide. After binding, it is first retrogradely transported to the Golgi and then to the endoplasmic reticulum (ER). Using a multivalent peptide library approach, we identified a tetravalent peptide that exhibits a high affinity for the Stx2 B‐ subunit pentamer (KD=0.13 µM) and markedly inhibits Stx2 cytotoxicity. The tetravalent peptide exerted its inhibitory effects by inducing aberrant cellular transport of Stx2. Although the tetravalent peptide/Stx2 complex was incorporated into cells and translocated to the Golgi, this process was followed by the effective degradation of Stx2 in an acidic compartment rather than by its transfer to the ER. This peptide thoroughly protected mice from a fatal dose of E. coli O157:H7 even when administered after an established infection. Thus, the multivalent peptide library approach enabled the identification of a peptide‐based Stx2 inhibitor that has remarkable therapeutic potency and appears to function by inducing aberrant cellular transport and degradation of Stx2.—Nishikawa, K., Watanabe, M., Kita, E., Igai, K., Omata, K., Yaffe, M. B., Natori, Y. A multivalent peptide library approach identifies a novel Shiga toxin inhibitor that induces aberrant cellular transport of the toxin. FASEB J. 20, E2077–E2086 (2006)

Yip1A regulates the COPI-independent retrograde transport from the Golgi complex to the ER

Yip1A, a mammalian homologue of yeast Yip1p, is a multi-spanning membrane protein that is considered to be involved in transport between the endoplasmic reticulum (ER) and the Golgi. However, the precise role of Yip1A in mammalian cells remains unclear. We show here that endogenous Yip1A is localized to the ER-Golgi intermediate compartment (ERGIC). Knockdown of Yip1A by RNAi did not induce morphological changes in the Golgi, ER, or ERGIC. By analyzing a number of intracellular transport pathways, we found that Yip1A knockdown delayed the transport of Shiga toxin from the Golgi to the ER, but did not affect the anterograde transport of VSVGts045. We also found that a recombinant protein that corresponded to the N-terminal domain of Yip1A inhibited the COPI-independent retrograde transport of GFP-tagged galactosyltransferase, GT-GFP, but not the COPI-dependent retrograde transport of p58/ERGIC53. Furthermore, we found that Yip1A knockdown resulted in the dissociation of Rab6 from the membranes. These results suggested that Yip1A has a role in COPI-independent retrograde transport from the Golgi to the ER and regulates the membrane recruitment of Rab6.

Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase

Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer’s disease.

A Peptide Library Approach Identifies a Specific Inhibitor for the ZAP-70 Protein Tyrosine Kinase

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.