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The vitamin K-dependent gamma-carboxylation system in the endoplasmic reticulum membrane responsible for gamma-carboxyglutamic acid modification of vitamin K-dependent proteins includes gamma-carboxylase and vitamin K 2,3-epoxide reductase (VKOR). An understanding of the mechanism by which this system works at the molecular level has been hampered by the difficulty of identifying VKOR involved in warfarin sensitive reduction of vitamin K 2,3-epoxide to reduced vitamin K(1)H(2), the gamma-carboxylase cofactor. Identification and cloning of VKORC1, a proposed subunit of a larger VKOR enzyme complex, have provided opportunities for new experimental approaches aimed at understanding the vitamin K-dependent gamma-carboxylation system. In this work we have engineered stably transfected baby hamster kidney cells containing gamma-carboxylase and VKORC1 cDNA constructs, respectively, and stably double transfected cells with the gamma-carboxylase and the VKORC1 cDNA constructs in a bicistronic vector. All engineered cells showed increased activities of the enzymes encoded by the cDNAs. However increased activity of the gamma-carboxylation system, where VKOR provides the reduced vitamin K(1)H(2) cofactor, was measured only in cells transfected with VKORC1 and the double transfected cells. The results show that VKOR is the rate-limiting step in the gamma-carboxylation system and demonstrate successful engineering of cells containing a recombinant vitamin K-dependent gamma-carboxylation system with enhanced capacity for gamma-carboxyglutamic acid modification. The proposed thioredoxin-like (132)CXXC(135) redox center in VKORC1 was tested by expressing the VKORC1 mutants Cys(132)/Ser and Cys(135)/Ser in BHK cells. Both of the expressed mutant proteins were inactive supporting the existence of a CXXC redox center in VKOR.

Being one of the key kinases downstream of Toll-like receptors, IRAK1 has initially thought to be responsible for NFkappaB activation. Yet IRAK1 knock-out mice still exhibit NFkappaB activation upon lipopolysaccharide (LPS) challenge, suggesting that IRAK1 may play other un-characterized function. In this report, we show that IRAK1 is mainly involved in Stat3 activation and subsequent interleukin-10 (IL-10) gene expression. Splenocytes from IRAK1-deficient mice fail to exhibit LPS-induced Stat3 serine phosphorylation and IL-10 gene expression yet still maintain normal IL-1beta gene expression upon LPS challenge. Mechanistically, we observe that IRAK1 modification upon LPS challenge leads to its modification, nuclear distribution, and interaction with Stat3. IRAK1 can directly use Stat3 as a substrate and cause Stat3 serine 727 phosphorylation. In addition, nuclear IRAK1 binds directly with IL-10 promoter in vivo upon LPS treatment. Atherosclerosis patients usually have elevated serum IL-10 levels. We document here that IRAK1 is constitutively modified and localized in the nucleus in the peripheral blood mononuclear cells from atherosclerosis patients. These observations reveal the mechanism for the novel role of IRAK1 in the complex Toll-like receptor signaling network and indicate that IRAK1 regulation may be intimately linked with the pathogenesis and/or resolution of atherosclerosis.

The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.

Vitronectin (VN) is an adhesive glycoprotein with roles in the complement, coagulation, and immune systems. Many of the functions of VN are mediated by a glycosaminoglycan binding site, near its carboxyl-terminal end. In this paper, we show that the highly sulfated glycosaminoglycans (GAGs), dextran sulfate, pentosan polysulfate, and fucoidan effectively augment [14C]putrescine incorporation into VN and cross-linking of VN into high molecular multimers by guinea pig liver transglutaminase (TG). Other GAGs including heparin, low molecular weight heparin, dermatan sulfate, keratan sulfate, and the nonsulfated dextrans were ineffective in accelerating these reactions. Dextran sulfate of average molecular mass 500 kDa was more effective than dextran sulfate of average molecular mass 5 kDa, supporting a template mechanism of action of the GAGs, in which VN molecules align on the GAG in a conformation suitable for cross-linking. The VN multimers catalyzed by TG retained functional activity in binding [3H]heparin, platelets, and plasminogen activator inhibitor type-1 (PAI-1). [3H]Heparin bound selectively to the 65-kDa monomeric band of VN and to the multimers derived from this band. PAI-1, however, bound equally to both the 75- and 65-kDa monomeric forms of VN, suggesting that the PAI-1 binding site on VN is distinct from the GAG binding site. The interaction of GAGs with the TG-catalyzed cross-linking of VN may facilitate studies of VN structure-function relationships.