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.

[Partial research progress of GGCX pathogenic variation associated phenotypes].

γ-glutamyl carboxylase (GGCX), also known as vitamin K-dependent glutamyl carboxylase, catalyzes the posttranslational modification of specific glutamate residues in vitamin K-dependent proteins (VKDPs), and participates multiple biological functions including blood coagulation, bone metabolism, vascular calcification, and cell proliferation. It has been reported originally that GGCX pathogenic variation causes blood coagulation deficiency, which is called as vitamin K-dependent coagulation factor deficiency 1 (VKCFD1). Recently, it has been found that GGCX gene variation results in multiple clinical phenotypes, including dermatological, ophthalmological, skeletal or cardiac abnormalities. Among them, dermatological phenotype is the most common, which is known as pseudoxanthoma elasticum-like syndrome. This paper has reviewed the GGCX pathogenic variation associated phenotypes, in order to increase the recognition of GGCX-related genetic diseases and to help its diagnosis and treatment.

Effect of prepropeptide replacement on γ-carboxylation and activity of recombinant coagulation factor IX.

Based on observations indicating that the γ-carboxylase enzyme has a lower affinity for the protein C (PC) propeptide and that the γ-carboxylase region in the PC propeptide has a higher net charge, expression of recombinant chimeric factor IX (FIX) equipped with the PC propeptide was studied. The prepropeptide of FIX was replaced with that of PC by SOEing PCR and after cloning, recombinant pMT-prepro PC/FIX was transfected into insect Drosophila S2 cells. The expression and activity of expressed FIX were analyzed employing antigen and activity analyses 72 h of post-induction with copper. Higher secretion (1.2 fold) and activity (1.6 fold) levels were observed for chimeric prepro- PC/FIX in relation to wild-type FIX. Furthermore, after barium citrate precipitation, the evaluation of fully γ-carboxylated FIX indicated that more than 51% of the total FIX produced with the PC prepropeptide was fully γ-carboxylated, representing a substantial improvement (twofold) over a system employing the native FIX propeptide in which 25% of the protein is fully γ-carboxylated. The data illustrated that the expression of FIX using the PC propeptide led to much higher fully γ-carboxylated material, which is preferred to FIX constructs tolerating the sequence for the native FIX propeptide expressed in heterologous S2 systems.

GGCX variants leading to biallelic deficiency to γ-carboxylate GRP cause skin laxity in VKCFD1 patients.

γ-Glutamyl carboxylase (GGCX) catalyzes the γ-carboxylation of 15 different vitamin K dependent (VKD) proteins. Pathogenic variants in GGCX cause a rare hereditary bleeding disorder called Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1). In addition to bleedings, some VKCFD1 patients develop skin laxity and skeletal dysmorphologies. However, the pathophysiological mechanisms underlying these non-hemorrhagic phenotypes remain elusive. Therefore, we have analyzed 20 pathogenic GGCX variants on their ability to γ-carboxylate six non-hemostatic VKD proteins in an in vitro assay, where GGCX variants were expressed in GGCX-/- cells and levels of γ-carboxylated co-expressed VKD proteins were detected by a functional ELISA. We observed that GGCX variants causing markedly reduced γ-carboxylation of Gla rich protein (GRP) in vitro were reported in patients with skin laxity. Reduced levels of γ-carboxylated Matrix gla protein (MGP) are not exclusive for causing skeletal dysmorphologies in VKCFD1 patients. In silico docking of vitamin K hydroquinone on a GGCX model revealed a binding site, which was validated by in vitro assays. GGCX variants affecting this site result in disability to γ-carboxylate VKD proteins and hence are involved in the most severe phenotypes. This genotype-phenotype analysis will help to understand the development of non-hemorrhagic phenotypes and hence improve treatment in VKCFD1 patients.

Biotransformation of Benzoate to 2,4,6-Trihydroxybenzophenone by Engineered Escherichia coli.

The synthesis of natural products by E. coli is a challenging alternative method of environmentally friendly minimization of hazardous waste. Here, we establish a recombinant E. coli capable of transforming sodium benzoate into 2,4,6-trihydroxybenzophenone (2,4,6-TriHB), the intermediate of benzophenones and xanthones derivatives, based on the coexpression of benzoate-CoA ligase from Rhodopseudomonas palustris (BadA) and benzophenone synthase from Garcinia mangostana (GmBPS). It was found that the engineered E. coli accepted benzoate as the leading substrate for the formation of benzoyl CoA by the function of BadA and subsequently condensed, with the endogenous malonyl CoA by the catalytic function of BPS, into 2,4,6-TriHB. This metabolite was excreted into the culture medium and was detected by the high-resolution LC-ESI-QTOF-MS/MS. The structure was elucidated by in silico tools: Sirius 4.5 combined with CSI FingerID web service. The results suggested the potential of the new artificial pathway in E. coli to successfully catalyze the transformation of sodium benzoate into 2,4,6-TriHB. This system will lead to further syntheses of other benzophenone derivatives via the addition of various genes to catalyze for functional groups.

Functional Analysis of the PCCA and PCCB Gene Variants Predicted to Affect Splicing.

It is estimated that up to one-third of all variants causing inherited diseases affect splicing; however, their deleterious effects and roles in disease pathogenesis are often not fully characterized. Given their prevalence and the development of various antisense-based splice-modulating approaches, pathogenic splicing variants have become an important object of genomic medicine. To improve the accuracy of variant interpretation in public mutation repositories, we applied the minigene splicing assay to study the effects of 24 variants that were predicted to affect normal splicing in the genes associated with propionic acidemia (PA)-PCCA and PCCB. As a result, 13 variants (including one missense and two synonymous variants) demonstrated a significant alteration of splicing with the predicted deleterious effect at the protein level and were characterized as spliceogenic loss-of-function variants. The analysis of the available data for the studied variants and application of the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) guidelines allowed us to precisely classify five of the variants and change the pathogenic status of nine. Using the example of the PA genes, we demonstrated the utility of the minigene splicing assay in the fast and effective assessment of the spliceogenic effect for identified variants and highlight the necessity of their standardized classification.

Vitamin K-Dependent γ-Glutamyl Carboxylase in Sertoli Cells Is Essential for Male Fertility in Mice.

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

The crystal structure of benzophenone synthase from Garcinia mangostana L. pericarps reveals the basis for substrate specificity and catalysis.

Benzophenone synthase (BPS) catalyzes the production of 2,4,6-trihydroxybenzophenone via the condensation of benzoyl-CoA and three units of malonyl-CoA. The biosynthetic pathway proceeds with the formation of the prenylated xanthone α-mangostin from 2,4,6-trihydroxybenzophenone. Structural elucidation was performed to gain a better understanding of the structural basis of the function of Garcinia mangostana L. (mangosteen) BPS (GmBPS). The structure reveals the common core consisting of a five-layer αßαßα fold as found in other type III polyketide synthase enzymes. The three residues Met264, Tyr266 and Gly339 are proposed to have a significant impact on the substrate-binding specificity of the active site. Crystallographic and docking studies indicate why benzoyl-CoA is preferred over 4-coumaroyl-CoA as the substrate for GmBPS. Met264 and Tyr266 in GmBPS are properly oriented for accommodation of the 2,4,6-trihydroxybenzophenone product but not of naringenin. Gly339 offers a minimal steric hindrance to accommodate the extended substrate. Moreover, the structural arrangement of Thr133 provides the elongation activity and consequently facilitates extension of the polyketide chain. In addition to its impact on the substrate selectivity, Ala257 expands the horizontal cavity and might serve to facilitate the initiation/cyclization reaction. The detailed structure of GmBPS explains its catalytic function, facilitating further structure-based engineering to alter its substrate specificity and obtain the desired products.

Structural Basis for Enzymatic Off-Loading of Hybrid Polyketides by Dieckmann Condensation.

While several bioactive natural products that contain tetramate or pyridone heterocycles have been described, information on the enzymology underpinning these functionalities has been limited. Here we biochemically characterize an off-loading Dieckmann cyclase, NcmC, that installs the tetramate headgroup in nocamycin, a hybrid polyketide/nonribosomal peptide natural product. Crystal structures of the enzyme (1.6 Å) and its covalent complex with the epoxide cerulenin (1.6 Å) guide additional structure-based mutagenesis and product-profile analyses. Our results offer mechanistic insights into how the conserved thioesterase-like scaffold has been adapted to perform a new chemical reaction, namely, heterocyclization. Additional bioinformatics combined with docking and modeling identifies likely candidates for heterocycle formation in underexplored gene clusters and uncovers a modular basis of substrate recognition by the two subdomains of these Dieckmann cyclases.

Enzymatic Plasticity Inspired by the Diterpene Cyclase CotB2.

Enzymatic plasticity, as a modern term referring to the functional conversion of an enzyme, is significant for enzymatic activity redesign. The bacterial diterpene cyclase CotB2 is a typical plastic enzyme by which its native form precisely conducts a chemical reaction while its mutants diversify the catalytic functions drastically. Many efforts have been made to disclose the mysteries of CotB2 enzyme catalysis. However, the catalytic details and regulatory mechanism toward the precise chemo- and stereoselectivity are still elusive. In this work, multiscale simulations are employed to illuminate the biocyclization mechanisms of the linear substrate into the final product cyclooctat-9-en-7-ol with a 5-8-5 fused ring scaffold, and the derailment products arising from the premature quenching of reactive carbocation intermediates are also discussed. The two major regulatory factors, local electrostatic stabilization effects from aromatic residues or polar residue in pocket and global features of active site including pocket-contour and pocket-hydrophobicity, are responsible for the enzymatic plasticity of CotB2. Further comparative studies of representative Euphorbiaceae and fungal diterpene cyclase (RcCS and PaFS) show a correlation between pocket plasticity and product diversity, which inspires a tentative enzyme product prediction and the rational diterpene cyclases' reengineering in the future.

Vitamin K in Vertebrates' Reproduction: Further Puzzling Pieces of Evidence from Teleost Fish Species.

Vitamin K (VK) is a fat-soluble vitamin that vertebrates have to acquire from the diet, since they are not able to de novo synthesize it. VK has been historically known to be required for the control of blood coagulation, and more recently, bone development and homeostasis. Our understanding of the VK metabolism and the VK-related molecular pathways has been also increased, and the two main VK-related pathways-the pregnane X receptor (PXR) transactivation and the co-factor role on the γ-glutamyl carboxylation of the VK dependent proteins-have been thoroughly investigated during the last decades. Although several studies evidenced how VK may have a broader VK biological function than previously thought, including the reproduction, little is known about the specific molecular pathways. In vertebrates, sex differentiation and gametogenesis are tightly regulated processes through a highly complex molecular, cellular and tissue crosstalk. Here, VK metabolism and related pathways, as well as how gametogenesis might be impacted by VK nutritional status, will be reviewed. Critical knowledge gaps and future perspectives on how the different VK-related pathways come into play on vertebrate's reproduction will be identified and proposed. The present review will pave the research progress to warrant a successful reproductive status through VK nutritional interventions as well as towards the establishment of reliable biomarkers for determining proper nutritional VK status in vertebrates.

Experimental Model of Subclinical Vitamin K Deficiency.

INTRODUCTION: Vitamin K (VK) is a co-factor in the post-translational gamma glutamic carboxylation of Gla-proteins. VK-dependent coagulation factors are carboxylated in the liver by VK1. Osteocalcin and Matrix-Gla protein (MGP) are carboxylated in extrahepatic tissues by VK2. A model of VK deficiency would be suitable for studying extrahepatic Gla-proteins provided that severe bleeding is prevented. AIM: The aim of this work was to adapt an established protocol of vascular calcification by warfarin-induced inactivation of MGP as a calcification inhibitor, in an attempt to create a broader state of subclinical VK deficiency and to verify its safety. MATERIALS AND METHODS: Two consecutive experiments, each lasting 4 weeks, were required to modify the dosing schedule of warfa-rin and VK1 and to adapt it to the Wistar rats used. The original high doses of warfarin used initially had to be halved and the protective dose of VK1 to be doubled, in order to avoid treatment-induced hemorrhagic deaths. The second experiment aimed to confirm the efficacy and safety of the modified doses. To verify the VK deficiency, blood vessels were examined histologically for calcium deposits and serum osteocalcin levels were mea-sured. RESULTS: The original dosing schedule induced VK deficiency, manifested by arterial calcifications and dramatic changes in carboxyl-ated and uncarboxylated osteocalcin. The modified dosing regimen caused similar vascular calcification and no bleeding. CONCLUSION: The modified protocol of carefully balanced warfarin and VK1 doses is an effective and safe way to induce subclinical VK deficiency that can be implemented to investigate VK-dependent proteins like osteocalcin.

Improvement of the recombinant human coagulation factor IX expression by co-expression of a novel transcript of Drosophila γ carboxylase in a human cell line.

OBJECTIVE: Mammalian cells as the main host for production of human proteins are incapable of complete γ-carboxylation of over-expressed Vitamin K Dependent (VKD) proteins. The Drosophila γ-glutamyl carboxylase (DγC) has been shown to be more efficient than its human counterpart in γ-carboxylation of human substrates, in vitro. Considering the Drosophila γ-carboxylase (DγC) efficiency, in comparison with its human counterpart, for recognition and γ-carboxylation of a human substrate in vitro, we were determined to study the effect of the DγC on the hFIX expression in a mammalian cell line. With this aim, we examined co-expression of the DγC with the hFIX, in a human cell line. RESULTS: While the co-expression of a complete DγC cDNA reduced the hFIX expression, a truncated form of DγC could improve both the expression level (up to 1211 ng/106 cells/ml on the 4th day of post-transfection) and carboxylation of the expressed hFIX, significantly (p < 0.009). CONCLUSIONS: Our findings provided evidences for potential of a partial fragment of the DγC for improvement of the γ-carboxylation of a human substrate in a mammalian cell. Our experimental data, in accordance with in silico analysis suggested that the DγC C-terminal fragment, with the advantage of a Kozak-like element has the potential of being expressed as a separate internal translation unit, to generate a peptide with appropriate γ-carboxylase activity.

Uncovering the chemistry of C-C bond formation in C-nucleoside biosynthesis: crystal structure of a C-glycoside synthase/PRPP complex.

The enzyme ForT catalyzes C-C bond formation between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and 4-amino-1H-pyrazole-3,5-dicarboxylate to make a key intermediate in the biosynthesis of formycin A 5'-phosphate by Streptomyces kaniharaensis. We report the 2.5 Å resolution structure of the ForT/PRPP complex and locate active site residues critical for PRPP recognition and catalysis.

Homology Modeling and Virtual Screening of Proteins Related to PXE and PXE-like Diseases: Insights for Overlapping Metabolites.

BACKGROUND: The molecular etiology of Pseudoxanthoma Elasticum (PXE), an autosomal recessive connective tissue disorder, has become increasingly complex as not only mutations in the ABCC6, but also in ENPP1 and GGCX, can cause resembling phenotypes. METHODS: To get insights on the common pathway, the overlapping metabolites for these three proteins were predicted through 3D homology modeling and virtual screening. 3D homology models of ABCC6, ENPP1, and GGCX were generated by the MODELLER program, which were further validated using RAMPAGE and ERRAT servers. Substrate binding sites of ABCC6 were predicted using blind docking of reported in vitro substrates. RESULTS: Virtual screening against the substrate binding site of ABCC6 using metabolites listed in Human Metabolome Databases (HMDB) revealed the best possible substrate of ABCC6. Those listed metabolites were further docked against predicted substrate binding sites of GGCX and ENPP1. Molecular docking and virtual screening revealed a list of 133 overlapping metabolites of these three proteins. Most of them are Phosphatidylinositol (PI), Phosphatidylserine (PS), Diacylglycerol (DAG), phosphatidic acid, oleanolic acid metabolites and were found to have links with calcification. CONCLUSION: These predicted overlapping metabolites may give novel insights for searching common pathomechanism for PXE and PXE-like diseases.

AccR, a TetR Family Transcriptional Repressor, Coordinates Short-Chain Acyl Coenzyme A Homeostasis in Streptomyces avermitilis.

Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are the most common extender units for the biosynthesis of fatty acids and polyketides in Streptomyces, an industrially important producer of polyketides. Carboxylation of acetyl- and propionyl-CoAs is an essential source of malonyl- and methylmalonyl-CoAs; therefore, acyl-CoA carboxylases (ACCases) play key roles in primary and secondary metabolism. The regulation of the expression of ACCases in Streptomyces spp. has not been investigated previously. We characterized a TetR family transcriptional repressor, AccR, that mediates intracellular acetyl-, propionyl-, methylcrotonyl-, malonyl-, and methylmalonyl-CoA levels by controlling the transcription of genes that encode the main ACCase and enzymes associated with branched-chain amino acid metabolism in S. avermitilis AccR bound to a 16-nucleotide palindromic binding motif (GTTAA-N6-TTAAC) in promoter regions and repressed the transcription of the accD1A1-hmgL-fadE4 operon, echA8, echA9, and fadE2, which are involved in the production and assimilation of acetyl- and propionyl-CoAs. Methylcrotonyl-, propionyl-, and acetyl-CoAs acted as effectors to release AccR from its target DNA, resulting in enhanced transcription of target genes by derepression. The affinity of methylcrotonyl- and propionyl-CoAs to AccR was stronger than that of acetyl-CoA. Deletion of accR resulted in increased concentrations of short-chain acyl-CoAs (acetyl-, propionyl-, malonyl-, and methylmalonyl-CoAs), leading to enhanced avermectin production. Avermectin production was increased by 14.5% in an accR deletion mutant of the industrial high-yield strain S. avermitilis A8. Our findings clarify the regulatory mechanisms that maintain the homeostasis of short-chain acyl-CoAs in StreptomycesIMPORTANCE Acyl-CoA carboxylases play key roles in primary and secondary metabolism. However, the regulation of ACCase genes transcription in Streptomyces spp. remains unclear. Here, we demonstrated that AccR responded to intracellular acetyl-, propionyl-, and methylcrotonyl-CoA availability and mediated transcription of the genes related to production and assimilation of these compounds in S. avermitilis When intracellular concentrations of these compounds are low, AccR binds to target genes and represses their transcription, resulting in low production of malonyl- and methylmalonyl-CoAs. When intracellular acetyl-, propionyl-, and methylcrotonyl-CoA concentrations are high, these compounds bind to AccR to dissociate AccR from target DNA, promoting the conversion of these compounds to malonyl- and methylmalonyl-CoAs. This investigation revealed how AccR coordinates short-chain acyl-CoA homeostasis in Streptomyces.

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.

Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study.

Vitamin K-dependent carboxylation is a post-translational modification essential for the biological function of coagulation factors. Defects in carboxylation are mainly associated with bleeding disorders. With the discovery of new vitamin K-dependent proteins, the importance of carboxylation now encompasses vascular calcification, bone metabolism, and other important physiological processes. Our current knowledge of carboxylation, however, comes mainly from in vitro studies carried out under artificial conditions, which have a limited usefulness in understanding the carboxylation of vitamin K-dependent proteins in native conditions. Using a recently established mammalian cell-based assay, we studied the carboxylation of coagulation factors in a cellular environment. Our results show that the coagulation factor's propeptide controls substrate binding and product releasing during carboxylation, and the propeptide of factor IX appears to have the optimal affinity for efficient carboxylation. Additionally, non-conserved residues in the propeptide play an important role in carboxylation. A cell-based functional study of naturally occurring mutations in the propeptide successfully interpreted the clinical phenotype of warfarin's hypersensitivity during anticoagulation therapy in patients with these mutations. Unlike results obtained from in vitro studies, results from our cell-based study indicate that although the propeptide of osteocalcin cannot direct the carboxylation of the coagulation factor, it is required for the efficient carboxylation of osteocalcin. This suggests that the coagulation factors may have a different mechanism of carboxylation from osteocalcin. Together, results from this study provide insight into efficiently controlling one physiological process, such as coagulation without affecting the other, like bone metabolism.

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.