The YCR079w gene confers a rapamycin-resistant function and encodes the sixth type 2C protein phosphatase in Saccharomyces cerevisiae

Type 2C protein phosphatase (PP2C) is a monomeric enzyme and requires Mg(2+) or Mn(2+) for its activity. Up to now, seven PP2C-like genes have been identified in the genome of Saccharomyces cerevisiae. However, the protein encoded by the sixth PP2C-like gene, YCR079w, has not been demonstrated to have PP2C activity. In this study, we show that YCR079w confers a rapamycin-resistant function in yeast cells, and we also demonstrate that the YCR079w-encoded protein exhibits characteristics of a typical PP2C. Therefore, YCR079w encodes the sixth PP2C, PTC6, in budding yeast.     

Shotgun glycopeptide capture approach coupled with mass spectrometry for comprehensive glycoproteomics

We present a robust and general shotgun glycoproteomics approach to comprehensively profile glycoproteins in complex biological mixtures. In this approach, glycopeptides derived from glycoproteins are enriched by selective capture onto a solid support using hydrazide chemistry followed by enzymatic release of the peptides and subsequent analysis by tandem mass spectrometry. The approach was validated using standard protein mixtures that resulted in a close to 100% capture efficiency. Our capture approach was then applied to microsomal fractions of the cisplatin-resistant ovarian cancer cell line IGROV-1/CP. With a Protein Prophet probability value greater than 0.9, we identified a total of 302 proteins with an average protein identification rate of 136 +/- 19 (n = 4) in a single linear quadrupole ion trap (LTQ) mass spectrometer nano-LC-MS experiment and a selectivity of 91 +/- 1.6% (n = 4) for the N-linked glycoconsensus sequence. Our method has several advantages. 1) Digestion of proteins initially into peptides improves the solubility of large membrane proteins and exposes all of the glycosylation sites to ensure equal accessibility to capture reagents. 2) Capturing glycosylated peptides can effectively reduce sample complexity and at the same time increase the confidence of MS-based protein identifications (more potential peptide identifications per protein). 3) The utility of sodium sulfite as a quencher in our capture approach to replace the solid phase extraction step in an earlier glycoprotein chemical capture approach for removing excess sodium periodate allows the overall capture procedure to be completed in a single vessel. This improvement minimizes sample loss, increases sensitivity, and makes our protocol amenable for high throughput implementation, a feature that is essential for biomarker identification and validation of a large number of clinical samples. 4) The approach is demonstrated here on the analysis of N-linked glycopeptides; however, it can be applied equally well to O-glycoprotein analysis.     

Identification and characterization of a functional Candida albicans homolog of the Saccharomyces cerevisiae TCO89 gene

As one of the components of target of rapamycin complex 1 (TORC1), ScTco89p is involved in rapamycin sensitivity and cellular integrity in Saccharomyces cerevisiae. Here we provide evidence showing that deletion of ScTCO89 causes yeast cells to be hypersensitive to salt stress in a high osmolarity glycerol pathway-independent fashion. In addition, we have identified and characterized a functional Candida albicans homolog (CaTCO89) of ScTCO89, which encodes a protein of 708 amino acids that shows overall 15% identity with ScTco89p at the amino acid level. However, CaTCO89 could complement the functions of ScTCO89 in rapamycin sensitivity, salt tolerance, and cellular integrity. Candida albicans cells disrupted for CaTCO89 are also sensitive to rapamycin and lithium salt, but not susceptible to challenges to cellular integrity.     

Generation of pigs with humanized type Ⅱ collagen by precise human COL2A1 gene knock-in

<正>Osteoarthritis (OA) is the most common chronic disease, characterized by progressive cartilage breakdown, subchondral bone sclerosis, and aberrant bone outgrowth (Yucesoy et al., 2015; Hussain et al., 2016). OA is one of the leading causes of cartilage damage. Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life. Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important rol...     

Loop CD20/CD19 CAR-T cells eradicate B-cell malignancies efficiently

CD19 chimeric antigen receptor(CAR) T cells have shown robust efficacy in relapsed and refractory acute lymphoblastic leukemia(R/R ALL), but compromising result in chronic lymphoblastic leukemia(CLL) and non-Hodgkin’s lymphoma(NHL).CD19-relapse and the lack of CAR-T cell persistence which result in treatment failure are considerable obstacles to overcome.CAR-T targeting CD20 is an option for salvaging CD19 CAR-T failure. Previous studies have established variant structures of bispecific CAR-T wh...     

The patterns and participants of parental histone recycling during DNA replication in Saccharomyces cerevisiae

Epigenetic information carried by histone modifications not only reflects the state of gene expression, but also participates in the maintenance of chromatin states and the regulation of gene expression. Recycling of parental histones to daughter chromatin after DNA replication is vital to mitotic inheritance of epigenetic information and the maintenance of cell identity,because the locus-specific modifications of the parental histones need to be maintained. To assess the precision of parental h...     

Machine learning-aided scoring of synthesis difficulties for designer chromosomes

Designer chromosomes are artificially synthesized chromosomes. Nowadays, these chromosomes have numerous applications ranging from medical research to the development of biofuels. However, some chromosome fragments can interfere with the chemical synthesis of designer chromosomes and eventually limit the widespread use of this technology. To address this issue,this study aimed to develop an interpretable machine learning framework to predict and quantify the synthesis difficulties of designer ch...     

Kinetics study of a selenium-containing ScFv catalytic antibody that mimics glutathione peroxidase

The steady-state kinetics study and some enzymatic characterization of a selenium-containing scFv catalytic antibody (Se-scFv2F3) were carried out. A novel reaction formula of this abzyme-catalyzed reaction was proposed and a rate equation was obtained according to the formula. The constants in the equation were compared with Dalziel's parameters and the exact meanings of these constants were analyzed. The obtained kinetics parameters from the kinetics study of Se-scFv2F3 were analyzed and compared with those of native glutathione peroxidase.     

Mechanism of integrin activation by disulfide bond reduction

Integrin alphaIIbbeta3 plays a pivotal role in hemostasis and thrombosis by mediating platelet adhesion and platelet aggregation. Integrin alphaIIbbeta3 contains an on/off switch that regulates its ligand binding affinity. The switch from "off" to "on" is commonly referred to as integrin activation. We recently identified a redox site within the extracellular domain of the platelet integrin alphaIIbbeta3 that exhibits many properties that one might expect of the on/off switch [Yan, B., and Smith, J. W. (2000) J. Biol. Chem. 275, 39964-39972]. Several independent reports show that reducing agents, such as dithiothreitol, can activate integrins. The objective of the present study was to determine if the effects of DTT can be attributed to a perturbation at the integrin redox site. Indeed, we find that DTT reduces two disulfide bonds within the integrin's cysteine-rich domain. Such bond reduction leads to global conformational changes within both alphaIIb and beta3 and the opening of the RGD and fibrinogen binding sites. These findings causally link the reduction of disulfide bonds within the integrin's redox site to transitions in the integrin's activation state.