Vitronectin is a substrate for transglutaminases

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.     

Ethanenitronate is a peroxide-dependent suicide substrate for catalase

We find ethanenitronate (formula; see text) to be a H2O2- (and peracetic acid-) dependent suicide substrate for bovine liver catalase (E) which converts E to Em, a modified form of the enzyme. The catalytic and suicide pathways are related to E, Em, Compound I, and Compound II according to the following scheme. (formula; see text) The catalytic cycle generates free radical products (EN.) which then participate in an O2-dependent chain reaction. Within experimental error the exclusive target for inactivation by EN- is Compound II. This partitions in the ratio (k4 = 1.2 M-1 s-1)/(k3 = 1.6 M-1 s-1) to generate Em and E, respectively. The species Em acquires 1 eq of 14C/ferriheme from [1-14C]ethanenitronate which is firmly (presumably covalently) affixed to the protein moiety. According to the standard H2O2 assay, Em is 7% as active catalytically as E. We regard inactivation as resulting from that fraction of EN. in the E...EN. complex which fails to diffuse from the complex because it is trapped by reaction with a neighboring amino acid residue to generate Em irreversibly. (formula; see text) This mechanism is identical to that deduced previously for suicide inactivation of horseradish peroxidase by alkane nitronates (Porter, D. J. T., and Bright, H. J. (1983) J. Biol. Chem. 258, 9913-9924) with the exception that EN. is trapped in that case by a methine carbon at the edge of the ferriheme rather than by the apoenzyme. The labeled residue in the catalase apoenzyme probably resides at or near the site of reduction of Compound II.     

Unstructured RNA is a substrate for tRNase Z

tRNase Z, which exists in almost all cells, is believed to be working primarily for tRNA 3' maturation. In Escherichia coli, however, the tRNase Z gene appears to be dispensable under normal growth conditions, and its physiological role is not clear. Here, to investigate a possibility that E. coli tRNase Z cleaves RNAs other than pre-tRNAs, we tested several unstructured RNAs for cleavage. Surprisingly, all these substrates were cleaved very efficiently at multiple sites by a recombinant E. coli enzyme in vitro. tRNase Zs from Bacillus subtilis and Thermotoga maritima also cleaved various unstructured RNAs. The E. coli and B. subtilis enzymes seem to have a tendency to cleave after cytidine or before uridine, while cleavage by the T. maritima enzyme inevitably occurred after CCA in addition to the other cleavages. Assays to determine optimal conditions indicated that metal ion requirements differ between B. subtilis and T. maritima tRNase Zs. There was no significant difference in the observed rate constant between unstructured RNA and pre-tRNA substrates, while the K(d) value of a tRNase Z/unstructured RNA complex was much higher than that of an enzyme/pre-tRNA complex. Furthermore, eukaryotic tRNase Zs from yeast, pig, and human cleaved unstructured RNA at multiple sites, but an archaeal tRNase Z from Pyrobaculum aerophilum did not.     

micF RNA is a substrate for RNase E

Abstract The speA gene encoding streptococcal erythrogenic toxin A (SPE A) from Streptococcus pyogenes bacteriophage T12 was overexpressed in Escherichia coli under the control of the T7 promoter. Since most of the expressed protein was found in the periplasmic space, an osmotic shock extraction with 0.5 M sucrose resulted in a highly enriched preparation of SPE A. An additional two-step purification employing high pressure liquid chromatography resulted in a purified SPE A protein.     

Pro-urokinase-type plasminogen activator is a substrate for hepsin

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys158-Ile159 (P1-P1') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (kcat/Km 4.8 x 10(5) m(-1) s(-1)) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 +/- 1.9 nm uPA h(-1), which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.     

Quercetin is a substrate for the transmembrane oxidoreductase Dcytb

Duodenal cytochrome b (Dcytb) is a transmembrane oxidoreductase protein found in apical membranes of duodenal enterocytes, as well as human erythrocytes, with the capacity to transport electrons donated by cytosolic ascorbate to extracellular electron receptors such as Fe(III), dehydroascorbate, or molecular O₂. We have investigated the capacity of the flavonoid quercetin to act as an electron donor for Dcytb in a manner similar to that of ascorbate by observing the reduction of extracellular Fe(III) to Fe(II) in either Madin–Darby canine kidney (MDCK) cells overexpressing Dcytb (Dcytb⁺) or Dcytb-null MDCK cells. In Dcytb⁺ cells there is a saturable increase in extracellular Fe(III) reduction in response to increasing intracellular quercetin concentrations (K_m = 6.53 ± 1.57 μM), in addition to a small linear response, whereas in Dcytb-null cells there is only a small linear increase in extracellular Fe(III) reduction. No extracellular Fe(III) reduction occurs in Dcytb-null cells when the cells are preloaded with ascorbate. Flavonoids such as quercetin at their physiological concentrations can therefore function as modulators of ferric reductases, enhancing the import of Fe(II) and also providing extracellular reducing potential.     

Thrombospondin is a substrate for blood coagulation factor XIIIa

Thrombospondin (TSP) is released from alpha granules of activated platelets, binds to platelet surfaces, and copolymerizes with fibrin. In the present experiments, we investigated the action of factor XIIIa (plasma transglutaminase) on TSP. Factor XIIIa catalyzed incorporation of [14C]putrescine into soluble TSP and ligation of TSP to itself and to fibrin intermediates. Proteolytic digestion of [14C]putrescine-labeled TSP with trypsin or thrombin yielded a labeled disulfide-bonded core of 90 or 120-130 kilodalton (kDa) subunits, labeled fragments of less than 10 kDa, and an unlabeled 30-kDa heparin-binding fragment, indicating the presence of multiple factor XIIIa reactive glutaminyl residues located in several domains of the molecule. TSP became ligated in fibrin clots formed from amidinated fibrinogen, i.e., fibrin that could not contribute lysyl residues to factor IIIa catalyzed cross-links. The disulfide-bonded core of TSP formed upon thrombin digestion copolymerized with fibrin as efficiently as intact TSP. However, a lower proportion of the disulfide-bonded core became ligated. These results indicate that TSP, both in clots and in solution, contributes glutaminyl and lysyl residues to factor XIIIa catalyzed ligation. Cross-linking may be important in stabilizing interactions among TSP, fibrinogen, or fibrin and other molecules in hemostatic plugs.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

Nitric oxide is a physiological substrate for mammalian peroxidases

We now show that NO serves as a substrate for multiple members of the mammalian peroxidase superfamily under physiological conditions. Myeloperoxidase (MPO), eosinophil peroxidase, and lactoperoxidase all catalytically consumed NO in the presence of the co-substrate hydrogen peroxide (H(2)O(2)). Near identical rates of NO consumption by the peroxidases were observed in the presence versus absence of plasma levels of Cl(-). Although rates of NO consumption in buffer were accelerated in the presence of a superoxide-generating system, subsequent addition of catalytic levels of a model peroxidase, MPO, to NO-containing solutions resulted in the rapid acceleration of NO consumption. The interaction between NO and compounds I and II of MPO were further investigated during steady-state catalysis by stopped-flow kinetics. NO dramatically influenced the build-up, duration, and decay of steady-state levels of compound II, the rate-limiting intermediate in the classic peroxidase cycle, in both the presence and absence of Cl(-). Collectively, these results suggest that peroxidases may function as a catalytic sink for NO at sites of inflammation, influencing its bioavailability. They also support the potential existence of a complex and interdependent relationship between NO levels and the modulation of steady-state catalysis by peroxidases in vivo.     

KIBRA is a novel substrate for protein kinase Czeta

WW domain-containing proteins are found in all eukaryotic cells and they are involved in the regulation of a wide variety of cellular functions. We recently identified the neuronal protein KIBRA as novel member of this family of signal transducers. In this report, we describe the identification of protein kinase C (PKC) zeta as a KIBRA-interacting protein. PKCzeta is known to play an important role in synaptic plasticity and memory formation but its specific targets are not well known. Our studies presented here revealed that KIBRA is a novel substrate for PKCzeta and suggest that PKCzeta phosphorylation may regulate the cellular function of KIBRA.     

Apolipoprotein A-IV is a novel substrate for matrix metalloproteinases

Screening of matrix metalloproteinase (MMP)-14 substrates in human plasma using a proteomics approach previously identified apolipoprotein A-IV (apoA-IV) as a novel substrate for MMP-14. Here, we show that among the tested MMPs, purified apoA-IV is most susceptible to cleavage by MMP-7, and that apoA-IV in plasma can be cleaved more efficiently by MMP-7 than MMP-14. Purified recombinant apoA-IV (44-kDa) was cleaved by MMP-7 into several fragments of 41, 32, 29, 27, 24, 22 and 19 kDa. N-terminal sequencing of the fragments identified two internal cleavage sites for MMP-7 in the apoA-IV sequence, between Glu(185) and Leu(186), and between Glu(262) and Leu(263). The cleavage of lipid-bound apoA-IV by MMP-7 was less efficient than that of lipid-free apoA-IV. Further, MMP-7-mediated cleavage of apoA-IV resulted in a rapid loss of its intrinsic anti-oxidant activity. Based on the fact that apoA-IV plays important roles in lipid metabolism and possesses anti-oxidant activity, we suggest that cleavage of lipid-free apoA-IV by MMP-7 has pathological implications in the development of hyperlipidemia and atherosclerosis.     

Fibronectin is a non-viral substrate for the HIV proteinase

The retrovirus encoded proteinase (PR) is required for the proper maturation of viral particles into infectious virus. The PR had been considered highly substrate specific, cleaving exclusively the viral gag and gag-pol protein precursor. It has recently been reported, however, that cytoskeleton and other cellular filament proteins can be cleaved by the HIV-1 PR. Here we have evidence that a cell-associated protein, the fibronectin (A-chain), is also cleaved in vitro specifically by this PR. The possibility of a cytotoxic role of the PR is conceivable.     

Phospholemman is a substrate for myotonic dystrophy protein kinase

The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I(Cl(PLM))) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I(Cl(PLM)) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I(Cl(PLM)). This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(-) PLM mutant in which all potential phosphorylation had been disabled by Ser --> Ala substitution. The biophysical characteristics of I(Cl(PLM)) were not changed by DMPK or by the phos(-) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I(Cl(PLM)) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.     

Apolipoprotein C-II is a novel substrate for matrix metalloproteinases

We previously reported an efficient proteomic approach to identify matrix metalloproteinase (MMP) substrates from complex protein mixture. Using the proteomic approach, apolipoprotein C-II (apoC-II), which is a cofactor of lipoprotein lipase (LPL) and a component of very-low density lipoprotein and chylomicron, has been identified as a putative MMP-14 substrate. Cleavage of apoC-II, with various MMPs, demonstrated that apoC-II is cleaved most efficiently by MMP-14, and also by MMP-7, among the tested MMPs. The 79-amino acid residue apoC-II was cleaved between Asn35 and Leu36 by MMP-14, and between Phe14 and Leu15 and between Asn35 and Leu36 by MMP-7. Cleavage of apoC-II by MMP-14 markedly decreased LPL activity and would thus impair hydrolysis of triglycerides in plasma and transfer of fatty acids to tissues. Our result suggests that cleavage of apoC-II by MMPs would be important for development of pathophysiological situations of apoC-II deficiency such as atherosclerosis.     

Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C

Vasodilator-stimulated phosphoprotein (VASP), an actin binding protein localized to areas of focal contacts, is a substrate for the cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP)-dependent protein kinases (PKA, PKG). In this study, we show that serum stimulation of vascular smooth muscle cells (SMCs) induces VASP phosphorylation on Ser157, in a mechanism not dependent on PKA or PKG. We tested the possibility that protein kinase C (PKC), a regulator of cytoskeletal function, is involved. PKC inhibition or down-regulation prevented serum-induced phosphorylation of VASP at Ser157 in rat vascular SMCs. Additionally, recombinant PKCalpha directly phosphorylated Ser157 on VASP. In summary, our data support the hypothesis that PKC phosphorylates VASP and mediates serum-induced VASP regulation.     

Hydrolase secretion is a consequence of membrane recycling

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.     

3-Aminotriazole is a substrate for lactoperoxidase but not for catalase

Rapid scan spectrophotometry has been applied to investigate the reaction of 3-aminotriazole with mammalian heme enzymes, represented by lactoperoxidase and bovine liver catalase. The results clearly indicate that 3-aminotriazole is a substrate for lactoperoxidase compounds I, II and III, but it does not convert catalase compound I to II under conditions favoring peroxidatic activity of the enzyme. The possible physiological significance of these findings is discussed.