Yeast as a tractable genetic system for functional studies of the insulin-degrading enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.     

Sequence imputation of HPV16 genomes for genetic association studies

Background

Human Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity.

Methods

A reference set of 62 HPV16 complete genome sequences was established and used to examine patterns of evolutionary relatedness amongst variants using a pairwise identity heatmap and HPV16 phylogeny. A BLAST-based algorithm was developed to impute complete genome data from partial sequence information using the reference database. To interrogate the oncogenic risk of determined and imputed HPV16 SNPs, odds-ratios for each SNP were calculated in a case-control viral genome-wide association study (VWAS) using biopsy confirmed high-grade cervix neoplasia and self-limited HPV16 infections from Guanacaste, Costa Rica.

Results

HPV16 variants display evolutionarily stable lineages that contain conserved diagnostic SNPs. The imputation algorithm indicated that an average of 97.5±1.03% of SNPs could be accurately imputed. The VWAS revealed specific HPV16 viral SNPs associated with variant lineages and elevated odds ratios; however, individual causal SNPs could not be distinguished with certainty due to the nature of HPV evolution.

Conclusions

Conserved and lineage-specific SNPs can be imputed with a high degree of accuracy from limited viral polymorphic data due to the lack of recombination and the stochastic mechanism of variation accumulation in the HPV genome. However, to determine the role of novel variants or non-lineage-specific SNPs by VWAS will require direct sequence analysis. The investigation of patterns of genetic variation and the identification of diagnostic SNPs for lineages of HPV16 variants provides a valuable resource for future studies of HPV16 pathogenicity.     

Novel functional assay for proteases and modulators

Proteases play a critical role in many cellular functions and have been an attractive therapeutic target due to their involvement in a number of disease processes. One prominent example is the secretases responsible for the generation of amyloid beta peptide, which is believed to be central for the development of Alzheimer's disease. It is therefore desirable to identify and characterize these proteases. We have developed a novel functional approach for identification of proteases and modulators by coupling the protease activity to caspase-mediated apoptosis. Here we show the proof of principle for this approach using beta-secretase as an example. We provide data showing that 1. A modified caspase-3 containing beta-secretase cleavage site induces apoptosis in 293T cells. 2. The modified caspase-3 induced apoptosis is correlated with the susceptibility of beta-secretase recognition sequence to beta-secretase. 3. In vivo beta-secretase competitors BACE2 and BACE2(D110A) prevent the modified caspase-3 induced cell death. Therefore, this approach can be a useful tool in studies of proteolytic cleavage provided only that the protease recognition sequence is known.     

A genome-wide comparison of the functional properties of rare and common genetic variants in humans

One of the longest running debates in evolutionary biology concerns the kind of genetic variation that is primarily responsible for phenotypic variation in species. Here, we address this question for humans specifically from the perspective of population allele frequency of variants across the complete genome, including both coding and noncoding regions. We establish simple criteria to assess the likelihood that variants are functional based on their genomic locations and then use whole-genome sequence data from 29 subjects of European origin to assess the relationship between the functional properties of variants and their population allele frequencies. We find that for all criteria used to assess the likelihood that a variant is functional, the rarer variants are significantly more likely to be functional than the more common variants. Strikingly, these patterns disappear when we focus on only those variants in which the major alleles are derived. These analyses indicate that the majority of the genetic variation in terms of phenotypic consequence may result from a mutation-selection balance, as opposed to balancing selection, and have direct relevance to the study of human disease.     

A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis.

Upon activation, cell surface death receptors, Fas/APO-1/CD95 and tumor necrosis factor receptor-1 (TNFR-1), are attached to cytosolic adaptor proteins, which in turn recruit caspase-8 (MACH/FLICE/Mch5) to activate the interleukin-1 beta-converting enzyme (ICE)/CED-3 family protease (caspase) cascade. However, it remains unknown whether these apoptotic proteases are generally involved in apoptosis triggered by other stimuli such as Myc and p53. In this study, we provide lines of evidence that a death protease cascade consisting of caspases and serine proteases plays an essential role in Myc-mediated apoptosis. When Rat-1 fibroblasts stably expressing either s-Myc or c-Myc were induced to undergo apoptosis by serum deprivation, a caspase-3 (CPP32)-like protease activity that cleaves a specific peptide substrate, Ac-DEVD-MCA, appeared in the cell lysates. Induction of s-Myc- and c-Myc-mediated apoptotic cell death was effectively prevented by caspase inhibitors such as Z-Asp-CH2-DCB and Ac-DEVD-CHO. Furthermore, exposing the cells to a serine protease inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), also significantly inhibited s-Myc- and c-Myc-mediated apoptosis and the appearance of the caspase-3-like protease activity in vivo. However, AEBSF did not directly inhibit caspase-3-like protease activity in the apoptotic cell lysates in vitro. Together, these results indicate that caspase-3-like proteases play a critical role in both s-Myc- and c-Myc-mediated apoptosis and that caspase-3-like proteases function downstream of the AEBSF-sensitive step in the signaling pathway of Myc-mediated apoptosis.     

稀有变异遗传关联性研究中常用负担检验方法比较

为比较稀有变异遗传关联研究中常用负担检验方法(CMC、WST、SUM及其扩展)在不同遗传情境下的统计性能,本文通过计算机模拟产生不同样本量、连锁不平衡(linkage disequilibrium, LD)参数、混杂非关联变异的个数和不同效应的关联变异等条件的稀有变异病例对照数据集,运用各种负担检验方法进行分析,分别计算各方法的一类错误和效能。结果表明,各方法一类错误均在0.05附近;当稀有变异效应方向一致时,除aSUM法外,LD参数越大、混杂非关联变异越少、各法效能越高;当效应方向不一致时,各法效能则显著降低。除强LD外,有方向考虑的方法效能均比无方向考虑的方法高,且样本量越大效能越高。负担检验的统计性能受效应大小和方向、噪音变异和连锁不平衡等多种因素影响。在实际应用中,在各类方法选择、确定集合单位,权重等时最好结合遗传变异的生物信息先验以提高研究效能。     

Molecular genetic system for regenerative studies using newts

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.     

A functional proteomics screen of proteases in colorectal carcinoma

BACKGROUND: Proteases facilitate several steps in cancer progression. To identify proteases most suitable for drug targeting, actual enzyme activity and not messenger RNA levels or immunoassay of protein is the ideal assay readout. MATERIALS AND METHODS: An automated microtiter plate assay format was modified to allow detection of all four major classes of proteases in tissue samples. Fifteen sets of colorectal carcinoma biopsies representing primary tumor, adjacent normal colon, and liver metastases were screened for protease activity. RESULTS: The major proteases detected were matrix metalloproteases (MMP9, MMP2, and MMP1), cathepsin B, cathepsin D, and the mast cell serine proteases, tryptase and chymase. Matrix metalloproteases were expressed at higher levels in the primary tumor than in adjacent normal tissue. The mast cell proteases, in contrast, were at very high levels in adjacent normal tissue, and not detectable in the metastases. Cathepsin B activity was significantly higher in the primary tumor, and highest in the metastases. The major proteases detected by activity assays were then localized in biopsy sections by immunohistochemistry. Mast cell proteases were abundant in adjacent normal tissue, because of infiltration of the lamina propria by mast cells. Matrix metalloproteases were localized to the tumor cells themselves; whereas, cathepsin B was predominantly expressed by macrophages at the leading edge of invading tumors. Although only low levels of urinary plasminogen activator were detected by direct enzyme assay, immunohistochemistry showed abundant protein within the tumor. CONCLUSIONS: This analysis, surveying all major classes of proteases by assays of activity rather than immunolocalization or in situ hybridization alone, serves to identify proteases whose activity is not completely balanced by endogenous inhibitors and which may be essential for tumor progression. These proteases are logical targets for initial efforts to produce low molecular weight protease inhibitors as potential chemotherapy.     

A flow cytometry-based screening system for directed evolution of proteases

Proteases are industrially important enzymes but often have to be improved for their catalytic efficiency and stabilities to suit applications. Flow cytometry screening technology based on in vitro compartmentalization in double emulsion had been developed and applied on directed evolution of paraoxonase and β-galactosidase. Further advancements of flow cytometry-based screening technologies will enable an ultra-high throughput of variants offering novel opportunities in directed enzyme evolution under high mutational loads. For the industrially important enzyme class of proteases, a first flow cytometry-based screening system for directed protease evolution has been developed based on an extracellular protease-deficient Bacillus subtilis strain (WB800N), a model protease (subtilisin Carlsberg), and a water-in-oil-in-water double-emulsion technology. B. subtilis WB800N cells are encapsulated in double emulsion with a fluorogenic substrate (rhodamine 110-containing peptide), allowing the screening of protease variants in femtoliter compartments at high throughput. The protease screening technology was validated by employing an epPCR mutant library with a high mutational load and screened for increased resistance toward the inhibitor antipain dihydrochloride. A variant (K127R, T237P, M239I, I269V, Y310F, I372V) with an improved relative resistance was isolated from a small population of active variants, validating the reported protease flow cytometry screening technology for increased inhibitor resistance.     

STAIRS: a new genetic resource for functional genomic studies of Arabidopsis

Many biologically and economically important traits in plants and animals are quantitative/multifactorial, being controlled by several quantitative trait loci (QTL). QTL are difficult to locate accurately by conventional methods using molecular markers in segregating populations, particularly for traits of low heritability or for QTL with small effects. In order to resolve this, large (often unrealistically large) populations are required. In this paper we present an alternative approach using a specially developed resource of lines that facilitate QTL location first to a particular chromosome, then to successively smaller regions within a chromosome (< or = 0.5 cM) by means of simple comparisons among a few lines. This resource consists of "Stepped Aligned Inbred Recombinant Strains" (STAIRS) plus single whole Chromosome Substitution Strains (CSSs). We explain the analytical power of STAIRS and illustrate their construction and use with Arabidopsis thaliana, although the principles could be applied to many organisms. We were able to locate flowering QTL at the top of chromosome 3 known to contain several potential candidate genes.     

Establishing a genetic system for ecological studies of Streptococcus oligofermentans

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.     

Yeast-based genetic system for functional analysis of poxvirus mRNA cap methyltransferase

Structural differences between poxvirus and human mRNA capping enzymes recommend cap formation as a target for antipoxviral drug discovery. Genetic and pharmacologic analysis of the poxvirus capping enzymes requires in vivo assays in which the readout depends on the capacity of the viral enzyme to catalyze cap synthesis. Here we have used the budding yeast Saccharomyces cerevisiae as a genetic model for the study of poxvirus cap guanine-N7 methyltransferase. The S. cerevisiae capping system consists of separate triphosphatase (Cet1), guanylyltransferase (Ceg1), and methyltransferase (Abd1) components. All three activities are essential for cell growth. We report that the methyltransferase domain of vaccinia virus capping enzyme (composed of catalytic vD1-C and stimulatory vD12 subunits) can function in lieu of yeast Abd1. Coexpression of both vaccinia virus subunits is required for complementation of the growth of abd1Delta cells. Previously described mutations of vD1-C and vD12 that eliminate or reduce methyltransferase activity in vitro either abolish abd1Delta complementation or elicit conditional growth defects. We have used the yeast complementation assay as the primary screen in a new round of alanine scanning of the catalytic subunit. We thereby identified several new amino acids that are critical for cap methylation activity in vivo. Studies of recombinant proteins show that the lethal vD1-C mutations do not preclude heterodimerization with vD12 but either eliminate or reduce cap methyltransferase activity in vitro.     

PROFICS: A bacterial selection system for directed evolution of proteases

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1′ residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1′ residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1′ tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.     

Structural and biochemical studies of retroviral proteases

Retroviral proteases form a unique subclass of the family of aspartic proteases. These homodimeric enzymes from a number of viral sources have by now been extensively characterized, both structurally and biochemically. The importance of such knowledge to the development of new drugs against AIDS has been, to a large extent, the driving force behind this progress. High-resolution structures are now available for enzymes from human immunodeficiency virus types 1 and 2, simian immunodeficiency virus, feline immunodeficiency virus, Rous sarcoma virus, and equine infectious anemia virus. In this review, structural and biochemical data for retroviral proteases are compared in order to analyze the similarities and differences between the enzymes from different sources and to enhance our understanding of their properties.     

Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.