A non-canonical vitamin K cycle is a potent ferroptosis suppressor.

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K-a group of naphthoquinones that includes menaquinone and phylloquinone3-confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

MCCC2 overexpression predicts poorer prognosis and promotes cell proliferation in colorectal cancer.

PURPOSES: Recently, Methylcrotonoyl-CoA carboxylase 2 (MCCC2) is reported to be involved in tumor formation and progression. However, MCCC2 has nerve been reported in colorectal cancer. In this study, we aimed to investigate the role of MCCC2 in colorectal cancer. METHODS: 118 colorectal cancer and matched adjacent normal tissues were enrolled in this study. The expression level of MCCC2 was measured by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The clinical significance of MCCC2 and its influence on cell proliferation was further analyzed. RESULTS: Results shown that the mRNA levels of MCCC2 in colorectal cancer tissues were significantly increased compared with those in normal tissues (P < .0001). MCCC2 high-expression was observed in 56.8% colorectal cancer tissues, which was significantly higher than those in normal controls (9.3%, P < .0001). MCCC2 high-expression correlated with tumor size, T stage, lymph node metastasis, distant metastasis, clinical stage and differentiation in colorectal cancer (P < .05). Moreover, MCCC2 high-expression predicted poorer prognosis and could be as an independent prognostic factor. In addition, MCCC2 knockdown significantly inhibited cell proliferation compared with these controls, while MCCC2 overexpression could reverse the effect. CONCLUSION: These data indicate MCCC2 overexpression promotes cell proliferation and predicts poorer prognosis in colorectal cancer.

Effect of GGCX on the differentiation function of osteoporosis bone marrow mesenchymal stem cells through regulating TGFß/smad signaling pathway.

OBJECTIVE: Osteoporosis (OP) has a high incidence and can be found in multiple age groups. The bone marrow mesenchymal stem cells (BMSCs) have the potential for self-renewal and multi-directional differentiation, which are often used for investigating the differentiation function of osteoporosis bone marrow mesenchymal stem cells. γ-glutamyl carboxylase (GGCX) is a carboxylase-related carboxylase and was observed to be abnormally expressed in osteoarthritis. However, the role and related mechanisms of GGCX in OP have not been fully elucidated. This work aimed to evaluate the effect of GGCX on the differentiation function of BMSCs. PATIENTS AND METHODS: Sprague-Dawley rats were randomly divided into the OP group prepared by ovariectomy and sham group. GGCX expression was tested by enzyme-linked immunosorbent assay (ELISA). BMSCs were isolated from OP rats and transfected with pcDNA-GGCX plasmids. BMSC proliferation was detected by tetrazolium salt colorimetry (MTT) assay. The osteogenic and adipogenic differentiation of BMSCs was analyzed by alizarin red staining and oil red O staining. The ALP activity was determined by alkaline phosphatase (ALP) activity colorimetric assay. Real time-PCR was used to test the expressions of osteogenesis-related genes RUNX2 and OPN mRNA. Western blot was adopted to assess the TGFß/smad signaling pathway activity. RESULTS: GGCX expression was significantly decreased in the serum of OP rats compared with the sham group (p < 0.05). The transfection of pcDNA-GGCX plasmid significantly promoted BMSC cell proliferation, increased calcified nodule formation, inhibited adipogenic differentiation, enhanced ALP activity, elevated RUNX2, and OPN mRNA expressions, and upregulated TGFß1, Smad2, and Smad7 expressions (p < 0.05). CONCLUSIONS: GGCX secretion is reduced in osteoporosis. GGCX can regulate osteoporosis via promoting the TGFß/smad signaling pathway, facilitating BMSCs osteogenic differentiation, and inhibiting BMSCs adipogenic differentiation.

A novel multidomain acyl-CoA carboxylase in Saccharopolyspora erythraea provides malonyl-CoA for de novo fatty acid biosynthesis.

Acetyl-CoA carboxylases (ACCs) are enzyme complexes generally composed of three catalytic domains and distributed in all organisms. In prokaryotes and plastids of most plants, these domains are encoded in distinct subunits forming heteromeric complexes. Distinctively, cytosolic ACCs from eukaryotes and plastids of graminaceous monocots, are organized in a single multidomain polypeptide. Until now, no multidomain ACCs had been discovered in bacteria. Here, we show that a putative multidomain ACC in Saccharopolyspora erythraea is encoded by the sace_4237 gene, representing the first prokaryotic ACC homodimeric multidomain complex described. The SACE_4237 complex has both acetyl-CoA and propionyl-CoA carboxylase activities. Importantly, we demonstrate that sace_4237 is essential for S. erythraea survival as determined by the construction of a sace_4237 conditional mutant. Altogether, our results show that this prokaryotic homodimeric multidomain ACC provides malonyl-CoA for de novo fatty acid biosynthesis. Furthermore, the data presented here suggests that evolution of these enzyme complexes, from single domain subunits to eukaryotic multidomain ACCs, occurred in bacteria through domain fusion.

Zinc alpha2 glycoprotein promotes browning in adipocytes.

Recent studies have highlighted recruiting and activating brite adipocytes in WAT (so-called "browning") would be an attractive anti-obesity strategy. Zinc alpha2 glycoprotein (ZAG) as an important adipokine, is reported to ameliorate glycolipid metabolism and lose body weight in obese mice. However whether the body reducing effect mediated by browning programme remains unclear. Here, we show that overexpression of ZAG in 3T3-L1 adipocytes enhanced expression of brown fat-specific markers (UCP-1, PRDM16 and CIDEA), mitochondrial biogenesis genes (PGC-1α, NRF-1/2 and mtTFA) and the key lipid metabolism lipases (ATGL, HSL, CPT1-A and p-acyl-CoA carboxylase). Additionally, those effects were dramaticlly abolished by H89/SB203580, revealing ZAG-induced browning depend on PKA and p38 MAPK signaling. Overall, our findings suggest that ZAG is a candidate therapeutic agent against obesity via induction of brown fat-like phenotype in white adipocytes.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

Effects of GGCX overexpression on anterior cruciate ligament transection-induced osteoarthritis in rabbits.

Effective therapeutic methods for osteoarthritis (OA) are lacking. γ­glutamyl carboxylase (GGCX) is a key enzyme that regulates carboxylation of cartilage matrix Gla protein (MGP). Whether GGCX overexpression protects against OA remains unknown. The aim of the present study was to explore the effects of GGCX overexpression on anterior cruciate ligament transection (ACLT)­induced OA and its mechanisms in Japanese white rabbits. ACLT surgery was used to establish an OA model in rabbits. A total of 48 rabbits were randomly divided into 4 groups: Sham, OA model + GGCX overexpression plasmid, OA model + saline and OA model + empty vector. The expression of uncarboxylated MGP (ucMGP), carboxylated MGP (cMGP), matrix metalloproteinase (MMP)­13, collagen type X, collagen type II, tumor necrosis factor (TNF)­α and interleukin (IL)­1ß were detected by ELISA, immunohistochemistry, reverse transcription­quantitative polymerase chain reaction and western blotting. Morphological changes to tibial cartilage were assessed by Giemsa and safranin O­fast green staining, respectively. Compared with the Sham control, GGCX expression was significantly decreased in the OA Model group. GGCX expression was increased by injection of a lentivirus­carried overexpression plasmid that encoded GGCX. GGCX overexpression ameliorated ATLC­induced damage in articular cartilage. OA Model rabbits exhibited significantly decreased expression levels of cMGP and collagen type II, and increased expression of ucMGP, collagen type X, MMP­13, IL­1ß and TNF­α. Notably, these expression levels were reversed by GGCX overexpression in OA Model rabbits. Results from the present study indicated that GGCX expression was decreased in OA Model rabbits, whereas overexpression of GGCX was able to promote carboxylation of MGP, reduce inflammation, decrease MMP­13 expression and regulate collagen expression. The results also indicated that GGCX may serve as a therapeutic target for OA.

[ANALYSIS OF γ-GLUTAMYL CARBOXYLASE GENE rs2592551 POLYMORPHISM ASSOCIATION WITH ISCHEMIC ATHEROTHROMBOTIC STROKE].

The results of γ-glutamyl carboxylase gene rs2592551 polymorphism determining in 170 patients with ischemic atherothrombotic stroke and 124 subjects without acute cerebrovascular disease (control group) have been evaluated. Obtained results revealed that rs2592551 polymorphism was related to ischemic stroke in Ukrainian population. The risk for this disease in patients with T/T genotype was higher than in major C-allele carriers (odds ratio (OR) = 3.117; 95% confidence interval (CI) = 1.016-9.566; P = 0.047). After dividing patients into subgroups, formed by the presence of certain risk factors for atherosclerosis, similar association has been established for women and non-smokers. At the same time, the heterozygous genotype (C/T) in females had significantly protective effect against ischemic stroke development when compared to C/C and T/T genotypes (OR = 0.460; 95% CI 0.213-0.994; P = 0.048). Statistical significance of these results persisted even after adjustment for age, body mass index, smoking and hypertension.

Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-glutamyl carboxylase activity than MK-4 both in vitro and in vivo.

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.

Methylcrotonoyl-CoA carboxylase 1 potentiates RLR-induced NF-κB signaling by targeting MAVS complex.

RNA virus infections are detected by the RIG-I family of receptors, which signal through the adaptor molecule mitochondrial antiviral signaling (MAVS). MAVS then recruits the adaptor's tumor necrosis factor receptor-associated factor (TRAF) 3 and TRAF6, which in turn activate IRF3 and NF-κB, respectively, to induce interferons (IFNs) and inflammatory responses. Here we show that the biotin-containing enzyme methylcrotonoyl-CoA carboxylase 1 (MCCC1) enhances virus-induced, MAVS-mediated IFN and inflammatory cytokine expression through the NF-κB signaling pathway. MCCC1 knockdown strongly inhibits induction of IFNs and inflammatory cytokines. Furthermore, MCCC1 shows extensive antiviral activity toward RNA viruses, including influenza A virus, human enterovirus 71, and vesicular stomatitis virus. Here, we have elucidated the mechanism underlying MCCC1-mediated inhibition of viral replication. MCCC1 interacts with MAVS and components of the MAVS signalosome and contributes to enhanced production of type I IFNs and pro-inflammatory cytokines by promoting phosphorylation of the IκB kinase (IKK) complex and NF-κB inhibitor-α (IκBα), as well as NF-κB nuclear translocation. This process leads to activation of IFNs and cytokine expression and subsequent activation of IFN-stimulated genes, including double-stranded RNA-dependent protein kinase PKR and myxovirus resistance protein 1. These findings demonstrate that MCCC1 plays an essential role in virus-triggered, MAVS-mediated activation of NF-κB signaling.

Structural and functional insights into enzymes of the vitamin K cycle.

Vitamin K-dependent proteins require carboxylation of certain glutamates for their biological functions. The enzymes involved in the vitamin K-dependent carboxylation include: gamma-glutamyl carboxylase (GGCX), vitamin K epoxide reductase (VKOR) and an as-yet-unidentified vitamin K reductase (VKR). Due to the hydrophobicity of vitamin K, these enzymes are likely to be integral membrane proteins that reside in the endoplasmic reticulum. Therefore, structure-function studies on these enzymes have been challenging, and some of the results are notably controversial. Patients with naturally occurring mutations in these enzymes, who mainly exhibit bleeding disorders or are resistant to oral anticoagulant treatment, provide valuable information for the functional study of the vitamin K cycle enzymes. In this review, we discuss: (i) the discovery of the enzymatic activities and gene identifications of the vitamin K cycle enzymes; (ii) the identification of their functionally important regions and their active site residues; (iii) the membrane topology studies of GGCX and VKOR; and (iv) the controversial issues regarding the structure and function studies of these enzymes, particularly, the membrane topology, the role of the conserved cysteines and the mechanism of active site regeneration of VKOR. We also discuss the possibility that a paralogous protein of VKOR, VKOR-like 1 (VKORL1), is involved in the vitamin K cycle, and the importance of and possible approaches for identifying the unknown VKR. Overall, we describe the accomplishments and the remaining questions in regard to the structure and function studies of the enzymes in the vitamin K cycle.

SH2 domain-containing inositol 5-phosphatase (SHIP2) regulates de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells.

Hepatic de-novo lipogenesis and production of triglyceride rich VLDL are regulated via the phosphoinositide 3-kinase cascade, however, the role of a negative regulator of this pathway, the SH2 domain-containing inositol 5-phosphatase (SHIP2) in this process, remains unknown. In the present study, we investigated the molecular link between SHIP2 expression and metabolic dyslipidemia using overexpression or suppression of SHIP2 gene in HepG2 cells. The results showed that overexpression of the wild type SHIP2 gene (SHIP2-WT) led to a higher total lipid content (28%) compared to control, whereas overexpression of the dominant negative SHIP2 gene (SHIP2-DN) reduced total lipid content in oleate treated cells by 40%. Overexpression of SHIP2-WT also led to a significant increase in both secretion of apoB100 containing lipoproteins and de-novo lipogenesis, as demonstrated by an enhancement in secreted apoB100 and MTP expression, increased intra and extracellular triglyceride levels and enhanced expression of lipogenic genes such as SREBP1c, FAS and ACC. On the other hand, overexpression of the SHIP2-DN gene prevented oleate-induced de-novo lipogenesis and secretion of apoB100 containing lipoproteins in HepG2 cells. Collectively, these findings suggest that SHIP2 expression level is a key determinant of hepatic lipogenesis and lipoprotein secretion, and its inhibition could be considered as a potential target for treatment of dyslipidemia.

The role of carbonic anhydrase in the pathogenesis of vascular calcification in humans.

Carbonic anhydrases are a group of isoenzymes that catalyze the reversible conversion of carbon dioxide into bicarbonate. They participate in a constellation of physiological processes in humans, including respiration, bone metabolism, and the formation of body fluids, including urine, bile, pancreatic juice, gastric secretion, saliva, aqueous humor, cerebrospinal fluid, and sweat. In addition, carbonic anhydrase may provide carbon dioxide/bicarbonate to carboxylation reactions that incorporate carbon dioxide to substrates. Several isoforms of carbonic anhydrase have been identified in humans, but their precise physiological role and the consequences of their dysfunction are mostly unknown. Carbonic anhydrase isoenzymes are involved in calcification processes in a number of biological systems, including the formation of calcareous spicules from sponges, the formation of shell in some animals, and the precipitation of calcium salts induced by several microorganisms, particularly urease-producing bacteria. In human tissues, carbonic anhydrase is implicated in calcification processes either directly by facilitating calcium carbonate deposition which in turn serves to facilitate calcium phosphate mineralization, or indirectly via its action upon γ-glutamyl-carboxylase, a carboxylase that enables the biological activation of proteins involved in calcification, such as matrix Gla protein, bone Gla protein, and Gla-rich protein. Carbonic anhydrase is implicated in calcification of human tissues, including bone and soft-tissue calcification in rheumatological disorders such as ankylosing spondylitis and dermatomyositis. Carbonic anhydrase may be also involved in bile and kidney stone formation and carcinoma-associated microcalcifications. The aim of this review is to evaluate the possible association between carbonic anhydrase isoenzymes and vascular calcification in humans.

Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase.

Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.

Inhibition of cytochrome P4502E1 by chlormethiazole attenuated acute ethanol-induced fatty liver.

Cytochrome P4502E1 (CYP2E1) has been demonstrated to play crucial roles in chronic ethanol-induced fatty liver, while its role in acute ethanol-induced fatty liver remains unclear. The current study was designed to evaluate the effects of chlormethiazole (CMZ), a specific inhibitor of CYP2E1, on acute ethanol-induced fatty liver, and to explore the mechanisms. Mice were pretreated with single dose of CMZ (50mg/kg body weight) by intraperitoneal injection or equal volume of saline, and then exposed to three doses of ethanol (5g/kg body weight, 25%, w/v) by gavage with 12h intervals. The mice were sacrificed at 4h after the last ethanol dosing. It was found that CMZ significantly attenuated acute ethanol-induced increase of the hepatic and serum triglyceride levels, and reduced fat droplets accumulation in mice liver. Acute ethanol-induced increase of the hepatic malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels (two biomarkers for oxidative stress) and decrease of glutathione (GSH) level was significantly suppressed by CMZ. CMZ also suppressed ethanol-induced decline of serum adiponectin level, but did not significantly affect the serum tumor necrosis factor-α (TNF-α) and ethanol levels. Furthermore, a significant decline of p62 protein level was observed in CMZ/ethanol group mice liver compared with that of the ethanol group mice. However, acute ethanol-induced increase of peroxisome proliferator-activated receptor α (PPAR-α) protein level was suppressed by CMZ, while the protein levels of sterol regulatory element-binding protein-1c (SREBP-1) and diacylglycerol acyltransferase 2 (DGAT2) were not significantly affected by ethanol or CMZ. Collectively, the results of the current study demonstrated that CMZ could effectively attenuate acute ethanol-induced fatty liver possibly by suppressing oxidative stress and adiponectin decline, and activating autophagy, which suggest that CYP2E1 might also play important roles in acute ethanol-induced fatty liver.

A conformational investigation of propeptide binding to the integral membrane protein γ-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry.

Gamma (γ)-glutamyl carboxylase (GGCX) is an integral membrane protein responsible for the post-translational catalytic conversion of select glutamic acid (Glu) residues to γ-carboxy glutamic acid (Gla) in vitamin K-dependent (VKD) proteins. Understanding the mechanism of carboxylation and the role of GGCX in the vitamin K cycle is of biological interest in the development of therapeutics for blood coagulation disorders. Historically, biophysical investigations and structural characterizations of GGCX have been limited due to complexities involving the availability of an appropriate model membrane system. In previous work, a hydrogen exchange mass spectrometry (HX MS) platform was developed to study the structural configuration of GGCX in a near-native nanodisc phospholipid environment. Here we have applied the nanodisc-HX MS approach to characterize specific domains of GGCX that exhibit structural rearrangements upon binding the high-affinity consensus propeptide (pCon; AVFLSREQANQVLQRRRR). pCon binding was shown to be specific for monomeric GGCX-nanodiscs and promoted enhanced structural stability to the nanodisc-integrated complex while maintaining catalytic activity in the presence of carboxylation co-substrates. Noteworthy modifications in HX of GGCX were prominently observed in GGCX peptides 491-507 and 395-401 upon pCon association, consistent with regions previously identified as sites for propeptide and glutamate binding. Several additional protein regions exhibited minor gains in solvent protection upon propeptide incorporation, providing evidence for a structural reorientation of the GGCX complex in association with VKD carboxylation. The results herein demonstrate that nanodisc-HX MS can be utilized to study molecular interactions of membrane-bound enzymes in the absence of a complete three-dimensional structure and to map dynamic rearrangements induced upon ligand binding.

Impaired vitamin K recycling in uremia is rescued by vitamin K supplementation.

In chronic kidney disease, vitamin K-dependent proteins, including the calcification inhibitor matrix Gla protein, are largely uncarboxylated indicating that functional vitamin K deficiency may contribute to uremic vascular calcification. Since the effects of uremia on the vitamin K cycle are unknown, we investigated the influence of uremia and vitamin K supplementation on the activity of the vitamin K cycle and extraosseous calcification. Uremia was induced in rats by an adenine-supplemented diet and vitamin K1 or K2 was administered over 4 and 7 weeks. After 4 weeks of adenine diet, the activity of the vitamin K cycle enzyme γ-carboxylase but not the activities of DT-diaphorase or vitamin K epoxide reductase were reduced. Serum levels of undercarboxylated matrix Gla protein increased, indicating functional vitamin K deficiency. There was no light microscopy-detectable calcification at this stage but chemically determined aortic and renal calcium content was increased. Vitamin K treatment reduced aortic and renal calcium content after 4 weeks. Seven weeks of uremia induced overt calcification in the aorta, heart, and kidneys; however, addition of vitamin K restored intrarenal γ-carboxylase activity and overstimulated it in the liver along with reducing heart and kidney calcification. Thus, uremic vitamin K deficiency may partially result from a reduction of the γ-carboxylase activity which possibly contributes to calcification. Pharmacological vitamin K supplementation restored the vitamin K cycle and slowed development of soft tissue calcification in experimental uremia.

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily.

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme-ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e(-70)) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.

Structure and function of biotin-dependent carboxylases.

Biotin-dependent carboxylases include acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC), 3-methylcrotonyl-CoA carboxylase (MCC), geranyl-CoA carboxylase, pyruvate carboxylase (PC), and urea carboxylase (UC). They contain biotin carboxylase (BC), carboxyltransferase (CT), and biotin-carboxyl carrier protein components. These enzymes are widely distributed in nature and have important functions in fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, polyketide biosynthesis, urea utilization, and other cellular processes. ACCs are also attractive targets for drug discovery against type 2 diabetes, obesity, cancer, microbial infections, and other diseases, and the plastid ACC of grasses is the target of action of three classes of commercial herbicides. Deficiencies in the activities of PCC, MCC, or PC are linked to serious diseases in humans. Our understanding of these enzymes has been greatly enhanced over the past few years by the crystal structures of the holoenzymes of PCC, MCC, PC, and UC. The structures reveal unanticipated features in the architectures of the holoenzymes, including the presence of previously unrecognized domains, and provide a molecular basis for understanding their catalytic mechanism as well as the large collection of disease-causing mutations in PCC, MCC, and PC. This review will summarize the recent advances in our knowledge on the structure and function of these important metabolic enzymes.

Des-γ-carboxyl prothrombin is associated with tumor angiogenesis in hepatocellular carcinoma.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a hypervascular tumor, and angiogenesis plays an important role in its development. Previously, we demonstrated that des-γ-carboxyl prothrombin (DCP) promotes both cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) by inducing the autophosphorylation of kinase insert domain receptor (KDR). In the present study, DCP-associated tumor angiogenesis was assessed by comparing hypovascular and common hypervascular HCC. METHODS: The solitary HCCs of 827 patients were classified into two groups according to the tumor density at the arterial phase of a dynamic computed tomography scan; the initial clinical data of patients with the hyper- and hypovascular types were compared. The HCC tissues from 95 tumors were analyzed by immunohistochemical staining for DCP and phosphorylated KDR, and intratumoral microvessel density (MVD) was analyzed to evaluate microvessel angiogenesis. RESULTS: The serum DCP levels (320 ± 3532 mAU/mL) and tumor size (18.4 ± 9.0 mm) of patients with hypervascular HCC were significantly greater than those with hypovascular HCC (38.7 ± 80 mAU/mL and 14.6 ± 5.2 mm, P < 0.001). Immunohistochemical analysis revealed that the expressions of DCP and phospho-KDR were significantly greater in hypervascular HCC (71.4% and 31.0%, respectively) than in hypovascular HCC (7.6% and 5.7%, respectively). Intratumoral MVD was significantly correlated with DCP (r = 0.48, P < 0.0001). CONCLUSIONS: des-γ-carboxyl prothrombin production is associated with tumor angiogenesis in HCC.