Γ-carboxyglutamic Acid Research Articles

Low expression of PRRG2 in kidney renal clear cell carcinoma: an immune infiltration-associated prognostic biomarker

ObjectiveThis study aims to explore the prognostic significance of Proline-rich γ-carboxyglutamic acid protein 2 (PRRG2) in Kidney Renal Clear Cell Carcinoma (KIRC), a prevalent and deadly cancer, and its association with immune cell infiltration, a key strategy in developing effective biomarkers.MethodsThe study meticulously elucidated the prognostic significance and potential role of PRRG2 in KIRC, correlating its expression with patient sex, age, metastasis, and pathological stage. Utilizing Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA), the involvement of PRRG2 in immune response was investigated. The association between PRRG2 expression and immune cell infiltration was also scrutinized. Ultimately, cellular and tissue identity were confirmed via immunohistochemical staining and quantitative real-time PCR.ResultsThe study elucidates a notable decrease in PRRG2 expression in KIRC patients, correlating with demographic factors, metastasis, and pathological staging, and portending an unfavorable prognosis. Bioinformatic analyses underscore PRRG2’s role in immune response, with its expression significantly tied to immune cell infiltration and marker expression.ConclusionPRRG2 may potentially impact prognosis in KIRC patients by regulating immune infiltration, thus rendering PRRG2 a promising candidate prognostic biomarker for KIRC-associated immune infiltration.

Plasma levels and urinary excretion of protein Z in patients with urolithiasis.

Protein Z (PZ) is a γ-carboxyglutamic acid protein present in plasma that is involved in blood coagulation. Detailed analysis of urinary stones from patients with urolithiasis has revealed that PZ is often found in urinary stones composed of calcium oxalate monohydrate. In this study, we compared blood and urinary PZ concentrations between healthy individuals and patients with urolithiasis. Plasma and urine were collected from healthy individuals and patients with urolithiasis who provided informed consent. PZ was detected as a urinary stone matrix protein in some of the patients. PZ was quantified by ELISA, creatinine was measured by the enzymatic method, and the total protein concentration was measured by the Bradford method. The plasma PZ level was 2.54 ± 1.02 μg/mL in healthy individuals and that in urolithiasis patients classified by stone history were from 1.16 ± 0.77 to 3.73 ± 1.09 μg/mL, which was not significantly different. The urinary excretion of PZ (PZ/creatinine) was also not different in patients with urolithiasis and in healthy individuals (from 54.1 ± 40.9 to 95.4 ± 69.4 ng/mg vs. 73.3 ± 36.0 ng/mg). A positive correlation was found between the plasma PZ level and creatinine-corrected urinary PZ concentration (r = 0.46). Both the plasma level and urinary excretion of PZ in urolithiasis patients were not significantly different with normal individuals. PZ detected in urinary stones as a matrix protein is thought to be incorporated into urinary stones regardless of blood and urine levels of PZ.

Anti-cancer Effect of Unique Cartilage Matrix-associated Protein in Breast Cancer Cells Depends on γ-Carboxylation.

Unique cartilage matrix-associated protein (UCMA), a recently discovered vitamin K-dependent protein (VKDP) with a large number of γ-carboxyglutamic acid (Gla) residues, is associated with ectopic calcifications. Although the function of VKDPs is related to their γ-carboxylation status, the carboxylation status of UCMA in breast cancer is still unknown. Here, we investigated the inhibitory effect of UCMA with differing γ-carboxylation status on breast cancer cell lines, such as MDA-MB-231, 4T1, and E0771 cells. Undercarboxylated UCMA (ucUCMA) was generated by mutating the γ-glutamyl carboxylase (GGCX) recognition sites. The ucUCMA and carboxylated UCMA (cUCMA) proteins were collected from culture media of HEK293-FT cells that had been transfected with mutated GGCX and wild-type UCMA expression plasmids, respectively. Boyden Transwell and colony formation assays were performed to evaluate cancer cell migration, invasion, and proliferation. Culture medium containing cUCMA protein inhibited the migration, invasion, and colony formation of MDA-MB-231 and 4T1 cells to a greater degree than medium containing ucUCMA protein. Significant reductions in the migration, invasion, and colony formation were also observed in cUCMA-treated E0771 cells compared to those in ucUCMA-treated cells. The inhibitory role of UCMA in breast cancer is closely related to its γ-carboxylation status. The results of this study may be a basis for the development of UCMA-based anti-cancer drugs.

Structural and mutational analyses of decarboxylated osteocalcin provide insight into its adiponectin-inducing activity.

An acidic environment in bone is essential for bone metabolism and the production of decarboxylated osteocalcin, which functions as a regulatory hormone of glucose metabolism. Here, we describe the high-resolution X-ray crystal structure of decarboxylated osteocalcin under acidic conditions. Decarboxylated osteocalcin at pH 2.0 retains the α-helix structure of native osteocalcin with three γ-carboxyglutamic acid residues at neutral pH. This implies that decarboxylated osteocalcin is stable under an acidic environment in bone. In addition, site-directed mutagenesis revealed that Glu17 and Glu21 are important for the adiponectin-inducing activity of decarboxylated osteocalcin. These findings suggest that the receptor of decarboxylated osteocalcin responds to the negative charge in helix 1 of osteocalcin.

Osteocalcin active center models: Electrochemical adsorption on platinum and quantum chemical analysis

An attempt was made to simulate the electrochemical behavior of the osteocalcin active site simple models on the platinum component of a bimetallic Ti/Pt implant. Dicarboxylic glutamic acid and dicarboxylic aspartic acid, and ethylmalonic acid as the third model successfully complementing the model range. The surface coverage of the platinum electrode during adsorption of model dicarboxylic acids was estimated using cyclic voltammetry and electrochemical impedance spectroscopy. All three compounds demonstrate similar trends in the surface coverage dependencies on the adsorption potential, acids' adsorption onto platinum surface is weak, and in none of cases studied amino group participates in adsorption on the platinum electrode surface. Using quantum chemical calculations, the most stable conformations of model compounds and the values of voids between adsorbed dicarboxylic acid molecules were obtained. Since all three models have a relatively low surface coverage and are participants in the outer sphere process of nonspecific adsorption on platinum, the residues of γ-carboxyglutamic acid present in osteocalcin will not be specifically adsorbed on the platinum component of the bimetallic implant, and the absence of interactions between it and the platinum electrode should have a positive effect on the dynamics of osteoregeneration.

Evaluation of the Level and Polymorphism of the Osteocalcin Gene in Patients with Rheumatoid Arthritis

The hormone is known as the bone protein γ-carboxyglutamic acid. The hormone belongs to the family of proteins that depend on vitamin K, as this vitamin is a catalyst in the formation of (γ-carboxylation) that determines its attraction to the bone matrix and calcium, and this contributes to the formation of bones. Moreover, the non-carboxylated form of the hormone that exerts hormonal properties and is also involved in glucose and energy metabolism, stimulates osteoblasts to secrete insulin directly and indirectly from the pancreas through the synthesis of glucagon-like-peptide 1 GLP-1 From the small intestine this enhances insulin sensitivity in fat cells and muscle cells. This property may have an effect on bone mineralization and may lead to a change in bone quality and an increased risk of fractures.
 The aim of the study is to evaluate the level of osteocalcin in patients with rheumatoid arthritis in comparison with the healthy ones and its relationship with the enzymes (GSH, CP, SOD), and to detect the gene encoding the hormone osteocalcin in the disease and to find the relationship between the hormone osteocalcin and vital variables.
 Blood samples were collected from (30) healthy people (15) males and (15) females, their ages ranged from (25-60) years and (30) patients from (19) males and (11) females in a hospital Salah Al-Din General and Al-Sharqat General Hospital, and the analyzes were measured in the central laboratory of the hospital, which were examined by the specialist doctor.
 This study showed that the levels of OC in the blood serum of people with rheumatoid arthritis is statistically higher compared to the healthy ones, and a significant decrease in the level of GSH for patients compared with the healthy ones, and a significant increase in the level of CP and the level of SOD for patients compared with the healthy ones, and there is a positive correlation between the level of OC and the level of CP And a negative relationship between OC level, GSH level and SOD level. It was revealed that there were significant differences at the level of probability (P<0.01) of repeating the allele for the OC gene between the group of patients and the healthy ones, where the value of the repeating of the mutated C allele increased and the level of the normal T allele decreased within the group of patients compared to the healthy ones.

Cleavage-Responsive Biofactory T Cells Suppress Infectious Diseases-Associated Hypercytokinemia.

Severe infectious diseases, such as coronavirus disease 2019 (COVID‐19), can induce hypercytokinemia and multiple organ failure. In spite of the growing demand for peptide therapeutics against infectious diseases, current small molecule‐based strategies still require frequent administration due to limited half‐life and enzymatic digestion in blood. To overcome this challenge, a strategy to continuously express multi‐level therapeutic peptide drugs on the surface of immune cells, is established. Here, chimeric T cells stably expressing therapeutic peptides are presented for treatment of severe infectious diseases. Using lentiviral system, T cells are engineered to express multi‐level therapeutic peptides with matrix metallopeptidases‐ (MMP‐) and tumor necrosis factor alpha converting enzyme‐ (TACE‐) responsive cleavage sites on the surface. The enzymatic cleavage releases γ‐carboxyglutamic acid of protein C (PC‐Gla) domain and thrombin receptor agonist peptide (TRAP), which activate endothelial protein C receptor (EPCR) and protease‐activated receptor‐1 (PAR‐1), respectively. These chimeric T cells prevent vascular damage in tissue‐engineered blood vessel and suppress hypercytokinemia and lung tissue damages in vivo, demonstrating promise for use of engineered T cells against sepsis and other infectious‐related diseases.

Possible Association Between Polymorphisms in ESR1, COL1A2, BGLAP, SPARC, VDR, and MMP2 Genes and Dental Fluorosis in a Population from an Endemic Region of West Bengal.

Dental fluorosis (DF) is the most prevalent form of fluorosis in India affecting millions of people all over the country. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), secreted protein acidic and cysteine-rich (SPARC), vitamin D receptor (VDR), and matrix metallopeptidase 2 (MMP2) genes play critical roles in bone metabolism, bone formation, mineral metabolism, and mineralization, variants in these genes could influence susceptibility to DF. The present study was aimed at evaluating the association between 15 single-nucleotide polymorphisms (SNPs) in the six candidate genes (namely, ESR1, COL1A2, BGLAP, SPARC, VDR, and MMP2) and DF among 132 individuals (case = 71 and control = 61) living in a fluoride endemic region of West Bengal, India. No statistically significant association with disease risk was found when the genotypes and allele frequencies of each of the 15 SNPs was analyzed individually using odd's ratio with 95% confidence interval. "CC" and "AG" haplotypes of the COL1A2 gene showed a borderline association with DF. The present study is the first in India to use multifactor dimensionality reduction (MDR) analysis for identifying gene-gene and gene-environment interactions in fluorosis. The biomarker of serum fluoride showed a significant association with the disease state among the 17 attributes (15 SNPs and 2 biomarkers of urine fluoride and serum fluoride) (P value = 0.011). The best model of MDR analysis with maximized testing accuracy involved two SNPs from the ESR1 gene (rs9340799 and rs2077647) and one SNP from BGLAP gene (rs1543294) (P value < 0.0001).

Chemical Profiling, Bioactivity Evaluation and the Discovery of a Novel Biopigment Produced by Penicillium purpurogenum CBS 113139.

Biobased pigments are environmentally friendly alternatives to synthetic variants with an increased market demand. Production of pigments via fermentation is a promising process, yet optimization of the production yield and rate is crucial. Herein, we evaluated the potential of Penicillium purpurogenum to produce biobased pigments. Optimum sugar concentration was 30 g/L and optimum C:N ratio was 36:1 resulting in the production of 4.1–4.5 AU (namely Pigment Complex A). Supplementation with ammonium nitrate resulted in the production of 4.1–4.9 AU (namely Pigment Complex B). Pigments showed excellent pH stability. The major biopigments in Pigment Complex A were N-threonyl-rubropunctamin or the acid form of PP-R (red pigment), N-GABA-PP-V (violet pigment), PP-O (orange pigment) and monascorubrin. In Pigment Complex B, a novel biopigment annotated as N-GLA-PP-V was identified. Its basic structure contains a polyketide azaphilone with the same carboxyl-monascorubramine base structure as PP-V (violet pigment) and γ-carboxyglutamic acid (GLA). The pigments were not cytotoxic up to 250 μg/mL.

Analysis of 180 Genetic Variants in a New Interactive FX Variant Database Reveals Novel Insights into FX Deficiency

Coagulation factor X (FX), often termed as Stuart–Prower factor, is a plasma glycoprotein composed of the γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor domains (EGF-1 and EGF-2), and the serine protease (SP) domain. FX plays a pivotal role in the coagulation cascade, activating thrombin to promote platelet plug formation and prevent excess blood loss. Genetic variants in FX disrupt coagulation and lead to FX or Stuart–Prower factor deficiency. To better understand the relationship between FX deficiency and disease severity, an interactive FX variant database has been set up at https://www.factorx-db.org , based on earlier web sites for the factor-XI and -IX coagulation proteins. To date (April 2021), we report 427 case reports on FX deficiency corresponding to 180 distinct F10 genetic variants. Of these, 149 are point variants (of which 128 are missense), 22 are deletions, 3 are insertions, and 6 are polymorphisms. FX variants are phenotypically classified as being type I or II. Type-I variants involve the simultaneous reduction of FX coagulant activity (FX:C) and FX antigen levels (FX:Ag), whereas type-II variants involve a reduction in FX:C with normal FX:Ag plasma levels. Both types of variants were distributed throughout the FXa protein structure. Analyses based on residue surface accessibilities showed the most damaging variants to occur at residues with low accessibilities. The interactive FX web database provides a novel easy-to-use resource for clinicians and scientists to improve the understanding of FX deficiency. Guidelines are provided for clinicians who wish to use the database for diagnostic purposes.

Structure of human factor VIIa-soluble tissue factor with calcium, magnesium and rubidium.

Coagulation factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (GLA) domain, two epidermal growth factor-like (EGF) domains and a protease domain. FVIIa binds three Mg2+ ions and four Ca2+ ions in the GLA domain, one Ca2+ ion in the EGF1 domain and one Ca2+ ion in the protease domain. Further, FVIIa contains an Na+ site in the protease domain. Since Na+ and water share the same number of electrons, Na+ sites in proteins are difficult to distinguish from waters in X-ray structures. Here, to verify the Na+ site in FVIIa, the structure of the FVIIa-soluble tissue factor (TF) complex was solved at 1.8 Å resolution containing Mg2+, Ca2+ and Rb+ ions. In this structure, Rb+ replaced two Ca2+ sites in the GLA domain and occupied three non-metal sites in the protease domain. However, Rb+ was not detected at the expected Na+ site. In kinetic experiments, Na+ increased the amidolytic activity of FVIIa towards the synthetic substrate S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) by ∼20-fold; however, in the presence of Ca2+, Na+ had a negligible effect. Ca2+ increased the hydrolytic activity of FVIIa towards S-2288 by ∼60-fold in the absence of Na+ and by ∼82-fold in the presence of Na+. In molecular-dynamics simulations, Na+ stabilized the two Na+-binding loops (the 184-loop and 220-loop) and the TF-binding region spanning residues 163-180. Ca2+ stabilized the Ca2+-binding loop (the 70-loop) and Na+-binding loops but not the TF-binding region. Na+ and Ca2+ together stabilized both the Na+-binding and Ca2+-binding loops and the TF-binding region. Previously, Rb+ has been used to define the Na+ site in thrombin; however, it was unsuccessful in detecting the Na+ site in FVIIa. A conceivable explanation for this observation is provided.

Perspective: Evidence before Enthusiasm—A Critical Review of the Potential Cardiovascular Benefits of Vitamin K

A protective role for vitamin K in cardiovascular disease (CVD), a leading cause of morbidity and mortality, has been proposed because vitamin K-dependent proteins, such as matrix Gla (γ-carboxyglutamic acid) protein (MGP), are present in vascular tissue. MGP functions as a vascular calcification inhibitor-but only when it is carboxylated, which requires vitamin K. There is more than one naturally occurring form of vitamin K. Phylloquinone (vitamin K1) is found in plant-based foods, whereas menaquinones (vitamin K2) are a class of vitamin K compounds found in animal-based and fermented foods. Phylloquinone and menaquinones are capable of carboxylating MGP and other vitamin K-dependent proteins. In rodent models, high intakes of either phylloquinone or menaquinone reduced vascular calcification. Evidence of the relative importance of phylloquinone and menaquinone to CVD in humans is limited and controversial. In some observational studies, higher dietary menaquinone intake, but not phylloquinone intake, was associated with less coronary artery calcification (a subclinical manifestation of CVD) and a lower risk for clinical CVD events. These findings have led to claims that menaquinones have unique cardiovascular health benefits compared with phylloquinone. However, this claim is not supported by the results of the limited number of intervention trials conducted to date. The purpose of this review is to evaluate the strengths and limitations of the available evidence regarding the role of vitamin K in vascular calcification, CVD, and mortality.

The impact of a key nutraceutical complex on chondrocyte cells and matrix Gla protein expression

Osteoarthritis (OA) is the most common type of arthritis among adults, which is associated with cartilage breakdown. Chondrocytes are the only type of cell in articular cartilage that secrete extracellular matrix (ECM). Therefore, to prevent and manage the occurrence of arthritic cartilage, it is important to consider approaches to increase the survival of chondrocyte cells. The production of reactive oxygen species (ROS), the release of pro-inflammatory cytokines (e.g. interleukin-1β (IL-1β)), and cartilage calcification play an imperative role in OA pathophysiology. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) is an enzyme that regulates the production of ROS in chondrocyte cells. ROS and cytokines lead to ECM degradation and chondrocyte apoptosis. Moreover, the matrix γ-carboxyglutamic acid (Gla) protein (MGP) acts as a decalcification agent in chondrocytes, which can inhibit the pathogenic mineralisation of cartilage. The correct balance of nutrients may potentially have a protective effect against OA. In the present study, the effect of essential nutrients on Nox4 activity in chondrocyte cells, chondrocyte viability, and MGP expression was investigated. Chondrocyte cells were pre-treated with the nutrients, and the cells were subsequently assessed for Nox4 activity using the chemiluminescence technique. It was found that the production of ROS significantly decreased after preincubation in the presence of nutrients in comparison to the control samples. Furthermore, it was found that the presence of nutraceuticals improved the viability of chondrocyte cells and the expression of MGP, which acts as a mineralisation inhibitor in the chondrocyte growth plate. The results of this study suggest that the correct balance of key nutrients at the molecular level plays an essential role in supporting chondrocyte cells and promoting cartilage decalcification.

Roles of vitamin K (phylloquinone and menaquinones) and related proteins (prothrombin, des-γ-carboxyprothrombin, and γ-glutamic acid) in healthy adults

The aim of the study was to evaluate the plasma levels of vitamin K (VK), prothrombin, des-γ-carboxyprothrombin (DCP) by ELISA with two types of monoclonal antibodies (MU-3 and 19B7), and urinary γ-carboxyglutamic acid (γ-Gla) in healthy adults (45 males and 45 females) and 11 patients with liver cirrhosis. VK and γ-Gla were analyzed by reverse-phase HPLC, prothrombin was determined using a method with a synthetic chromogenic substrate, and DCPs were analyzed by immunoaffinity chromatography and reverse-phase HPLC, and measured by ELISA. We were able to detect phylloquinone K (PK) and menaquinones MK-4, -5, -6, -7, -8 and -9, and we isolated several DCPs from plasma. Age had significant positive correlations with levels of urinary γ-Gla, plasma MK-7 and total VK, and VKDPs (prothrombin and DCP using MU-3 antibody) in the healthy adults. Significant positive correlations were also found between urinary γ-Gla and plasma levels of PK, MK-4, MK-7, and total VK and VKDPs (prothrombin and DCP using MU-3 antibody) in the healthy adults. γ-Gla and total VK levels were significantly higher in healthy adults than in patients with liver cirrhosis, and VK components, prothrombin, DCPs, and the MU-3/19B7 ratio showed a tendency to be higher in the healthy adults. We conclude that the status of VK and related proteins may reflect the latent increase in des-γ-carboxylation in healthy aging.

PRRG4 promotes breast cancer metastasis through the recruitment of NEDD4 and downregulation of Robo1.

Metastasis is responsible for the death of most breast cancer patients. Robo1 has been implicated as a tumor suppressor for various cancers including breast cancer. However, it is not well understood how Robo1 expression is regulated during tumorigenesis. In this study, we uncovered that the transmembrane proline rich γ-carboxyglutamic acid protein 4 (PRRG4) promotes breast cancer metastasis by downregulating Robo1. Analysis of mRNA expression data in The Cancer Genome Atlas and immunohistochemistry assay on breast tumor samples showed that PRRG4 expression was higher in breast tumors than in normal breast tissues. Experiments with PRRG4 knockdown and overexpression revealed that PRRG4 promoted migration and invasion of breast cancer cells, and enhanced metastasis in an experimental metastasis model. Mechanistically, we found that PRRG4 via its LPSY and PPPY motifs recruited the E3 ubiquitin ligase NEDD4, which induced ubiquitination and degradation of Robo1, thus contributing to migration and invasion of breast cancer cells. In addition, PRRG4 interacted with and enhanced protein tyrosine kinase Src and FAK activation. Overall, our data support a model that PRRG4 via NEDD4 downregulates the Robo1, resulting in the activation of Src and FAK and promoting breast cancer metastasis. PRRG4 may be a novel target for treating metastatic breast cancer.

Identification of Conomarphin Variants in the Conus eburneus Venom and the Effect of Sequence and PTM Variations on Conomarphin Conformations.

Marine cone snails belonging to the Conidae family make use of neuroactive peptides in their venom to capture prey. Here we report the proteome profile of the venom duct of Conus eburneus, a cone snail belonging to the Tesseliconus clade. Through tandem mass spectrometry and database searching against the C. eburneus transcriptome and the ConoServer database, we identified 24 unique conopeptide sequences in the venom duct. The majority of these peptides belong to the T and M gene superfamilies and are disulfide-bonded, with cysteine frameworks V, XIV, VI/VII, and III being the most abundant. All seven of the Cys-free peptides are conomarphin variants belonging to the M superfamily that eluted out as dominant peaks in the chromatogram. These conomarphins vary not only in amino acid residues in select positions along the backbone but also have one or more post-translational modifications (PTMs) such as proline hydroxylation, C-term amidation, and γ-carboxylation of glutamic acid. Using molecular dynamics simulations, the conomarphin variants were predicted to predominantly have hairpin-like or elongated structures in acidic pH. These two structures were found to have significant differences in electrostatic properties and the inclusion of PTMs seems to complement this disparity. The presence of polar PTMs (hydroxyproline and γ-carboxyglutamic acid) also appear to stabilize hydrogen bond networks in these conformations. Furthermore, these predicted structures are pH sensitive, becoming more spherical and compact at higher pH. The subtle conformational variations observed here might play an important role in the selection and binding of the peptides to their molecular targets.

Unique cartilage matrix-associated protein inhibits the migratory and invasive potential of triple-negative breast cancer

Triple-negative breast cancer (TNBC) that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is a breast cancer subtype with very aggressive metastasis and poor prognosis. Unique cartilage matrix-associated protein (UCMA) is a vitamin K-dependent protein (VKDP) with a high-density γ-carboxyglutamic acid (Gla) domain due to the action of vitamin K. UCMA promotes osteoblast differentiation and mineral deposition in bone and suppresses calcification in vessels. However, correlation between UCMA and TNBC is unknown. This study investigated the inhibitory effect of UCMA on TNBC cell in vitro migration, invasion, and colony formation in addition to in vivo tumorigenesis. Cell migration and invasion significantly decreased in Ucma-overexpressing MDA-MB-231 and 4T1 cells compared to the mock control cells. Also, colony formation and the number of colonies significantly decreased in Ucma-overexpressing MDA-MB-231 and 4T1 cells. These results indicate that UCMA significantly inhibits the migration, invasion, and colony formation of TNBC cells. In an in vivo xenograft mouse model, tumor growth significantly decreased in mice bearing Ucma-overexpressing TNBC cells compared to the mock control cells, indicating that UCMA reduced in vivo tumor growth, similar to the inhibitory role of UCMA in vitro. Survival analysis using publicly available database showed that high UCMA expression significantly correlated with favorable relapse-free survival in TNBC patients compared to those with the other VKDPs, matrix Gla protein (MGP) and osteocalcin (OCN). Collectively, this study suggests that UCMA is a promising new therapeutic agent for TNBC.

Evaluating the Association Between Dental Fluorosis and Polymorphisms in Bone Development and Mineralization Genes Among Population from a Fluoride Endemic Region of Eastern India.

Close to 12 million people in India are affected by more than the desirable level of fluoride in drinking water that could lead to dental, skeletal, and non-skeletal fluorosis. Dental fluorosis is a developmental defect that results in hypo-mineralization and pronounced porosity of enamel in the affected individuals. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), and secreted protein acidic and cysteine rich (SPARC) genes are involved in bone development and mineralization, polymorphisms in these genes could be determining factors in influencing the risk to fluorosis among the exposed individuals in fluoride endemic areas. A case-control study was carried out among a total of 87 individuals (case = 36, control = 51) to examine the association between selected polymorphisms in the ESR1, COL1A2, BGLAP, and SPARC genes and risk of dental fluorosis from a fluoride endemic region of Eastern India. Altogether, 10 single nucleotide polymorphisms (SNPs) in ESR1 (rs2234693, rs2228480, rs3798577, rs2077647, and rs9340799), COL1A2 (rs42524, rs412777), BGLAP (rs1800247), and SPARC (rs6579885, rs4958278) genes were genotyped through PCR-RFLP in these subjects. The association of the SNPs for disease risk estimation was measured by odds ratio with 95% confidence interval. The risk genotypes of none of the 10 SNPs showed statistically significant association with risk of dental fluorosis. Frequencies of the haplotypes in the intragenic SNPs of the ESR1, COL1A2, and SPARC genes did not reveal any statistically significant difference between the case and control groups. The present study is the first of its kind from India that has attempted to investigate possible involvement of genetic factors in influencing the risk to fluorosis among the population from a fluoride endemic region.

Impact of Glutamate Carboxylation in the Adsorption of the α-1 Domain of Osteocalcin to Hydroxyapatite and Titania.

One proposed mechanism of implant fouling is attributed to the nonspecific adsorption of non-collagenous bone matrix proteins (NCPs) onto a newly implanted interface. With the goal of capturing the fundamental mechanistic and thermodynamic forces that govern changes in these NCP recognition domains as a function of γ-carboxyglutamic acid (Gla) post-translational modification and surface chemistry, we probe the adsorption process of the most commonly occurring NCP, osteocalcin, onto a mineral and metal oxide surface. Here, we apply two enhanced sampling methods to independently probe the effects of post-translational modification and peptide structure on adsorption. First, well-tempered metadynamics was used to capture the binding of acetyl and N-methylamide capped glutamic acid and Gla single amino acids onto crystalline hydroxyapatite and titania model surfaces at physiological pH. Following this, parallel tempering metadynamics in the well-tempered ensemble (PTMetaD-WTE) was used to study adsorption of the α-1 domain of osteocalcin onto hydroxyapatite and titania. Simulations were performed for the α-1 domain of osteocalcin in both its fully decarboxylated (dOC) and fully carboxylated (OC) form. Our simulations find that increased charge density due to carboxylation results in increased interactions at the interface, and stronger adsorption of the single amino acids to both surfaces. Interestingly, the role of Gla in promoting compact and helical structure in the α-1 domain resulted in disparate binding modes at the two surfaces, which is attributed to differences in interfacial water behavior. Overall, this work provides a benchmark for understanding the mechanisms that drive adsorption of Gla-containing mineralizing proteins onto different surface chemistries.

Laminin G Domains of Protein S Bind and Inhibit Fixa

Background: Protein S (PS) is a vitamin K-dependent anticoagulant whose importance is distinguished by many hematological disorders in PS-deficient individuals. PS deficiency is associated with venous and arterial thrombosis, cerebral thrombophlebitis, myocardial infarction, ischemic stroke, and vascular calcification. The 75-kDa PS contains a γ-carboxyglutamic acid module, a thrombin sensitive region, four epidermal growth factor-like modules, and a sex hormone binding globulin-like region that consists of two laminin G domains (LG1 and LG2). PS has three different functions: (a) a cofactor for activated protein C; (b) a cofactor for tissue factor pathway inhibitor, and (c) a direct inhibitor of factor IXa (FIXa). Our laboratory discovered that PS binds FIXa with a dissociation constant (Kd) ~40 nM and inhibits FIXa's function. Currently, we are ascertaining the domain of PS responsible for binding FIXa. Result and Discussion : By using molecular modeling and biochemical assays, we demonstrated that the laminin G domains of PS are crucial for binding FIXa. However, we do not have any information regarding conformational or structural changes that occur when the LG domains and FIXa interact. To confirm our hypothesis, we used circular dichroism (CD), isothermal titration calorimetry (ITC) and biochemical assays to locate the region of the LG domains responsible for FIXa binding. CD spectroscopy gives details of protein secondary structure and conformational change. The conformational change was greatest for binding of LG1+2 to FIXa (LG1+2&amp;gt;LG1&amp;gt;LG2); thus, FIXa apparently has a greater affinity for LG1+2 than for either of the separate LG domains. ITC measures the stoichiometry and thermodynamics (binding affinity, enthalpy, entropy, and Gibbs free energy of protein-protein interaction. We observed that the binding affinity between LG1+2 and FIXa was higher than the affinity between either LG1 or LG2 and FIXa, with a trend of LG1+2&amp;gt;LG1&amp;gt;LG2. In addition, the differences in Gibbs free energy supported the same trend. The binding stoichiometry was 1:1 in all three cases, which implied that each LG construct binds one FIXa molecule. All three interactions were enthalpy driven, their binding was exothermic, and binding was favored by a negative enthalpy. In addition to the foregoing biophysical study, we performed biochemical assays to assess the activities of the LG domains. Thrombin generation assays were performed with PS-deficient plasma supplemented with each LG domain construct and with intact PS. The reactions were initiated with a low tissue factor concentration (0.4 pm) to selectively generate thrombin in the intrinsic pathway. With PS-deficient plasma, the amount of thrombin produced decreased from 255 nM to 155 nM on addition of LG1+2. This result was comparable to the effect of adding intact PS (148 nM thrombin produced). Overall, the structural studies and the plasma studies indicated that LG1+2 is required to mimic the function of PS in binding to and inhibiting FIXa. Similarly, we previously observed that the LG1+2 domain inhibited generation of FXa by FIXa (by ~55%), which was similar to the inhibition of FIXa by intact PS. We conclude that the LG1+2 domain mimics the function of PS in inhibiting FIXa. Future Direction: To exactly identify the region in the PS LG domains responsible for binding to FIXa, we have created multiple single and double amino acid substitutions in the LG domains. Currently, we are identifying particular PS mutants unable to bind FIXa to precisely map the FIXa interaction region in PS. Disclosures No relevant conflicts of interest to declare.