Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.     

The inhibitor profiling of the caspase family of proteases using substrate-derived peptide glyoxals

A series of substrate-based α-keto-β-aldehyde (glyoxal) sequences have been synthesised and evaluated as inhibitors of the caspase family of cysteine proteases. A number of potent inhibitor sequences have been identified. For example, a palmitic acid containing sequence pal-Tyr-Val-Ala-Asp-glyoxal was demonstrated to be an extremely effective inhibitor of caspase-1, inhibiting not only the action of the protease against synthetic fluorogenic substrates (K_i = 0.3 nM) but also blocking its processing of pro-interleukin-1beta (pro-IL-1β). In addition, the peptide Ac-Asp-Glu-Val-Asp-glyoxal, which is based on the consensus cleavage sequence for caspase-3, is a potent inhibitor of this protease (K_i = 0.26 nM) yet only functions as a comparatively modest inhibitor of caspase-1 (K_i = 451 nM). Potent inhibitor sequences were also identified for caspases-6 and -8. However, the degree of discrimination between the family members is limited. The ability of Ac-Asp-Glu-Val-Asp-glyoxal to block caspase-3 like activity in whole cells and to delay the development of apoptosis was assessed. When tested against caspase-3 like activity in cell lysates, Ac-Asp-Glu-Val-Asp-glyoxal displayed effective inhibition similar to that observed against recombinant caspase-3. Treatment of whole cells with this potent caspase-3 inhibitor was however, not sufficient to significantly stall the development of apoptosis in-vitro.     

Protein-encapsulated bio-nanocapsules production with ER membrane localization sequences

Bio-nanocapsules (BNCs) are hollow nanoparticles composed of the L protein of hepatitis B virus (HBV) surface antigen (HBsAg), which can specifically introduce genes and drugs into various kinds of target cells. Although the classic electroporation method has typically been used to introduce highly charged molecules such as DNA, it is rarely adopted for proteins due to its very low efficiency. In this study, a novel approach to the preparation of BNC was established whereby a target protein was pre-encapsulated during the course of nanoparticle formation. Briefly, because of the process of BNC formation in a budding manner on the endoplasmic reticulum (ER) membrane, the association of target proteins to the ER membrane with lipidation sequences (ER membrane localization sequences) could directly generate protein-encapsulating BNC in collaboration with co-expression of the L proteins. Since the membrane-localized proteins are automatically enveloped into BNCs during the budding event, this method can be protect the proteins and BNCs from damage caused by electroporation and obviate the need for laborious consideration to study the optimal conditions for protein encapsulation. This approach would be a useful method for encapsulating therapeutic candidate proteins into BNCs.     

METCAM/MUC18 augments migration, invasion, and tumorigenicity of human breast cancer SK-BR-3 cells

Previous research has identified METCAM/MUC18, an integral membrane cell adhesion molecule (CAM) in the Ig-like gene super-family, as a promoter or a suppressor in the development of human breast cancer by MCF7, MDA-MB-231, and MDA-MB-468. To resolve these conflicting results we have investigated the role of this CAM in the progression of the three aforementioned cell lines plus one additional human breast cancer cell line, SK-BR-3. We transfected the SK-BR-3 cells with human METCAM/MUC18 cDNA to obtain G418-resistant clones, which expressed different levels of the protein and which were used to test the effect of human METCAM/MUC18 expression on in vitro motility, invasiveness, anchorage-independent colony formation in soft agar, disorganized growth in a 3D basement membrane culture assay, and in vivo tumorigenesis in athymic nude mice. Enforced METCAM/MUC18 expression increased in vitro motility, invasiveness, and anchorage-independent colony formation of SK-BR-3 cells and favored disorganized growth of the cells in 3D basement membrane culture. Enforced expression also increased tumorigenicity and final tumor weights of SK-BR-3 clones/cells after subcutaneous injection of the cells under the left third nipple of female athymic nude mice. To understand the mechanisms, we also determined the expression of several downstream key effectors in the tumors. Tumor cells from METCAM/MUC18 expressing clones exhibited elevated expression of an anti-apoptotic and survival index (Bcl2), an aerobic glycolysis index (LDH-A), and pro-angiogenesis indexes (VEGF and VAGFR2). We concluded that human METCAM/MUC18 promotes the development of breast cancer cells by increasing an anti-apoptosis and survival pathway and augmenting aerobic glycolysis and angiogenesis.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.     

Enhanced activity and stability of cellobiase (β-glucosidase: EC 3.2.1.21) produced in the presence of 2-deoxy-d-glucose from the fungus Termitomyces clypeatus

Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-d-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett.2006, 28, 1773-1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. K_m and V_max of the purified enzyme were measured as 0.187 mM and 0.018 U mg⁻¹, respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 °C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 °C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The β-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.     

Heterologous expression of Aspen PTM3, a MADS box gene in cotton

The PTM3 gene of Aspen was ectopically expressed in cotton to explore the opportunity to introduce desirable agronomic traits with the potential to improve yield and modify the duration of the parent cotton variety. Sixty-seven transgenic cotton lines expressing Aspen PTM3 (MADS box) gene were developed. The transgenic cotton lines expressing PTM3 gene showed earliness of 4-15 days variations in flowering and maturity. The transgenic lines were confirmed by kanamycin leaf paint assay, GUS assay and PCR. Among 67 transgenic lines, the event-10 showed profuse branching, event-24 showed abnormal growth and the remaining events exhibited single erect phenotype. In addition, the event-24 produced no flower and this might be due to the positional effect of PTM3 gene integration. Southern blot analysis performed for event-10, 24 and 48 showed distinct single copy integrations of PTM3 gene cassette. GUS assay performed using various plant parts of event-10 showed constitutive expression of the transgene. In view of cotton breeding, among all the events, the event-10 was found to be phenotypically significant with earliness of 12 days in flowering and 15 days in maturity and yield enhancement of 27%. In addition, the event-10 showed no square dropping and allowed the plants to bear more number of bolls. Based on these results, event-10 was chosen to carryout the inheritance study of expressed characters in the progeny.     

Tannins present in Cichorium intybus enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adipocytes through PTP1B inhibition

Insulin resistance is a fundamental aspect for the etiology of non-insulin dependent diabetes mellitus (NIDDM) and has links with a wide array of secondary disorders including weight gain and obesity. The present study analyzes the effect of Cichorium intybus methanolic (CME) extract on glucose transport and adipocyte differentiation in 3T3-L1 cells by studying the radiolabelled glucose uptake and lipid accumulation assays, respectively. By performing detannification (CME/DT), the role of tannins present in CME on both the activities was evaluated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a dose-dependent response. Glucose uptake profile in the presence of PI3K and IRTK inhibitors (Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptake in inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME. Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression analysis revealed the unique behavior of CME and CME/DT. The duality exhibited by C. intybus through adipogenesis inhibition and PPARgamma up regulation is of interest. Current observation concludes that the activities possessed by C. intybus are highly desirable for the treatment of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1 adipocytes.