Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis.

Burkholderia stabilis strain FERMP-21014 secretes cholesterol esterase (BsChe), which is used in clinical settings to determine serum cholesterol levels. Previously, we constructed an expression plasmid with an endogenous constitutive promoter to enable the production of recombinant BsChe. In this study, we obtained one mutant strain with 13.1-fold higher BsChe activity than the wild type, using N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen. DNA-sequencing analysis revealed that the strain had lost chromosome 3 (∆Chr3), suggesting that the genes hindering BsChe production may be encoded on Chr3. We also identified common mutations in the functionally unknown BSFP_068720/30 genes in the top 10 active strains generated during transposon mutagenesis. As BSFP_068720/30/40 comprised an operon on Chr3, we created the BSFP_068720/30/40 disruption mutant and confirmed that each disruption mutant containing the expression plasmid exhibited ~ 16.1-fold higher BsChe activity than the wild type. Quantitative PCR showed that each disruption mutant and ΔChr3 had a ~ 9.4-fold higher plasmid copy number than the wild type. Structural prediction models indicate that BSFP_068730/40 is structurally homologous to the structural maintenance of chromosomes (SMC) protein MukBE, which is responsible for chromosome segregation during cell division. Conversely, BSFP_068720/30/40 disruption did not lead to a Chr3 drop-out. These results imply that BSFP_068720/30/40 is not a SMC protein but is involved in destabilizing foreign plasmids to prevent the influx of genetic information from the environment. In conclusion, the disruption of BSFP_068720/30/40 improved plasmid stability and copy number, resulting in exceptionally high BsChe production. KEY POINTS: • Disruption of BSFP_068720/30/40 enabled mass production of Burkholderia Che/Lip. • BSFP_068730/40 is an SMC protein homolog not involved in chromosome retention. • BSFP_068720/30/40 is likely responsible for the exclusion of exogenous plasmids.

Bacterial triacylglycerol lipase is a potential cholesterol esterase: Identification of a key determinant for sterol-binding specificity.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Production of recombinant extracellular cholesterol esterase using consistently active promoters in Burkholderia stabilis.

Burkholderia stabilis FERMP-21014 produces highly active cholesterol esterase in the presence of fatty acids. To develop an overexpression system for cholesterol esterase production, we carried out RNA sequencing analyses to screen strongly active promoters in FERMP-21014. Based on gene expression consistency analysis, we selected nine genes that were consistently expressed at high levels, following which we constructed expression vectors using their promoter sequences and achieved overproduction of extracellular cholesterol esterase under fatty acid-free conditions. Of the tested promoters, the promoter of BSFP_0720, which encodes the alkyl hydroperoxide reductase subunit AhpC, resulted in the highest cholesterol esterase activity (24.3 U mL-1). This activity level was 243-fold higher than that of the wild-type strain under fatty acid-free conditions. We confirmed that cholesterol esterase was secreted without excessive accumulation within the cells. The gene expression consistency analysis will be useful to screen promoters applicable to the overexpression of other industrially important enzymes.

Discovery of Novel Transient Receptor Potential Vanilloid 4 (TRPV4) Agonists as Regulators of Chondrogenic Differentiation: Identification of Quinazolin-4(3 H)-ones and in Vivo Studies on a Surgically Induced Rat Model of Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease characterized by joint destruction and loss of cartilage. There are many unmet needs in the treatment of OA and there are few promising candidates for disease-modifying OA drugs, particularly, anabolic agents. Here, we describe the identification of novel quinazolin-4(3 H)-one derivatives, which stimulate chondrocyte cartilage matrix production via TRPV4 and mitigate damaged articular cartilage. We successfully identified the water-soluble, highly potent quinazolin-4(3 H)-one derivative 36 and studied its intra-articular physicochemical profile to use in in vivo surgical OA model studies. Compound 36·HCl provided relief from OA damage in a rat medial meniscal tear (MT) model. Specifically, 36·HCl dose-dependently suppressed cartilage degradation and enhanced the messenger RNA expression of aggrecan and SOX9 in cartilage isolated from MT-operated rat knees compared with knees treated with vehicle. These results suggest that 36 induces anabolic changes in articular cartilage and consequently reduces OA progression.

Arid5a cooperates with Sox9 to stimulate chondrocyte-specific transcription.

SRY-box-containing gene 9 (Sox9) is an essential transcription factor in chondrocyte lineage determination and differentiation. Recent studies demonstrated that Sox9 controls the transcription of chondrocyte-specific genes in association with several other transcriptional regulators. To further understand the molecular mechanisms by which Sox9 influences transcriptional events during chondrocyte differentiation, we attempted to identify transcriptional partners of Sox9 and to examine their roles in chondrocyte differentiation. We isolated AT-rich interactive domain-containing protein 5a (Arid5a; also known as Mrf1) as an activator of the Col2a1 gene promoter from an ATDC5 cDNA library. Arid5a was highly expressed in cartilage and induced during chondrocyte differentiation. Furthermore, Arid5a physically interacted with Sox9 in nuclei and up-regulated the chondrocyte-specific action of Sox9. Overexpression of Arid5a stimulated chondrocyte differentiation in vitro and in an organ culture system. In contrast, Arid5a knockdown inhibited Col2a1 expression in chondrocytes. In addition, Arid5a binds directly to the promoter region of the Col2a1 gene and stimulates acetylation of histone 3 in the region. Our results suggest that Arid5a may directly interact with Sox9 and thereby enhance its chondrocyte-specific action.

The transcription factor Znf219 regulates chondrocyte differentiation by assembling a transcription factory with Sox9.

Sox9 is an essential transcription factor for chondrogenesis by regulating the expression of chondrogenic genes. However, its regulatory mechanism is not fully understood. To address this, we attempted to identify the transcriptional partners of Sox9 by screening the cDNA library of the chondrogenic cell line ATDC5 using the collagen 2α1 (Col2α1) gene promoter fused to a luciferase reporter gene. One of the positive clones encoded the Znf219 gene. Whole mount in situ hybridization experiments indicated that Znf219 mRNA was specifically expressed in the developing limb buds where Col2α1 and Sox9 were strongly expressed. Znf219 markedly enhanced the transcriptional activity of Sox9 on the Col2a1 gene promoter. In addition, Znf219 is physically associated with Sox9 and is colocalized with Sox9 in the nucleus. We also found that overexpression of Znf219 profoundly increased Sox9-induced mRNA expression of Col2a1, aggrecan and Col11a2. Consistently, knockdown of Znf219 decreased the Sox9-induced mRNA expression of these genes. Furthermore, a dominant-negative mutant Znf219 inhibited Bmp2-induced chondrocyte differentiation. Our results suggest that Znf219 plays an important role in the regulation of chondrocyte differentiation as a transcriptional partner of Sox9.

Paraspeckle protein p54nrb links Sox9-mediated transcription with RNA processing during chondrogenesis in mice.

The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, alpha 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54(nrb) suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.

Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation.

Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca2+/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.

Centaurin-alpha1 is a phosphatidylinositol 3-kinase-dependent activator of ERK1/2 mitogen-activated protein kinases.

Centaurin-alpha1 is known to be a phosphatidylinositol 3,4,5-triphosphate (PIP3)-binding protein that has two pleckstrin homology domains and a putative ADP ribosylation factor GTPase-activating protein domain. However, the physiological function of centaurin-alpha1 is still not understood. Here we have shown that transient expression of centaurin-alpha1 in COS-7 cells results in specific activation of ERK, and the activation is inhibited by co-expression of a dominant negative form of Ras. We have also found that a mutant form of centaurin-alpha1 that is unable to bind PIP3 fails to induce ERK activation and that a phosphatidylinositol 3-kinase inhibitor LY294002 inhibits centaurin-alpha1-dependent ERK activation. Furthermore, transient knockdown of centaurin-alpha1 by small interfering RNAs results in reduced ERK activation after epidermal growth factor stimulation in T-REx 293 cells. These results suggest that centaurin-alpha1 contributes to ERK activation in growth factor signaling, linking the PI3K pathway to the ERK mitogen-activated protein kinase pathway through its ability to interact with PIP3.

Large-scale identification and characterization of human genes that activate NF-kappaB and MAPK signaling pathways.

We have carried out a large-scale identification and characterization of human genes that activate the NF-kappaB and MARK signaling pathways. We constructed full-length cDNA libraries using the oligo-capping method and prepared an arrayed cDNA pool consisting of 150 000 cDNAs randomly isolated from the libraries. For analysis of the NF-kappaB signaling pathway, we introduced each of the cDNAs into human embryonic kidney 293 cells and examined whether it activated the transcription of a luciferase reporter gene driven by a promoter containing the consensus NF-kappaB binding sites. In total, we identified 299 cDNAs that activate the NF-kappaB pathway, and we classified them into 83 genes, including 30 characterized activator genes of the NF-kappaB pathway, 28 genes whose involvement in the NF-kappaB pathways have not been characterized and 25 novel genes. We then carried out a similar analysis for the identification of genes that activate the MARK pathway, utilizing the same cDNA resource. We assayed 145 000 cDNAs and identified 57 genes that activate the MARK pathway. Interestingly, 27 genes were overlapping between the NF-kappaB and the MAPK pathways, which may indicate that these genes play cross-talking roles between these two pathways.