Streamlining N-terminally anchored yeast surface display via structural insights into S. cerevisiae Pir proteins.

Surface display co-opts yeast's innate ability to embellish its cell wall with mannoproteins, thus converting the yeast's outer surface into a growing and self-sustaining catalyst. However, the efficient toolbox for converting the enzyme of interest into its surface-displayed isoform is currently lacking, especially if the isoform needs to be anchored to the cell wall near the isoform's N-terminus, e.g., through a short GPI-independent protein anchor. Aiming to advance such N-terminally anchored surface display, we employed in silico and machine-learning strategies to study the 3D structure, function, genomic organisation, and evolution of the Pir protein family, whose members evolved to covalently attach themselves near their N-terminus to the ß-1,3-glucan of the cell wall. Through the newly-gained insights, we rationally engineered 14 S. cerevisiae Hsp150 (Pir2)-based fusion proteins. We quantified their performance, uncovering guidelines for efficient yeast surface display while developing a construct that promoted a 2.5-fold more efficient display of a reporter protein than the full-length Hsp150. Moreover, we developed a Pir-tag, i.e., a peptide spanning only 4.5 kDa but promoting as efficient surface display of a reporter protein as the full-length Hsp150. These constructs fortify the existing surface display toolbox, allowing for a prompt and routine refitting of intracellular proteins into their N-terminally anchored isoforms.

Systematic Comparison of Cell Wall-Related Proteins of Different Yeasts.

Yeast cell walls have two major roles, to preserve physical integrity of the cell, and to ensure communication with surrounding molecules and cells. While the first function requires evolutionary conserved polysaccharide network synthesis, the second needs to be flexible and provide adaptability to different habitats and lifestyles. In this study, the comparative in silico analysis of proteins required for cell wall biosynthesis and functions containing 187 proteins of 92 different yeasts was performed in order to assess which proteins were broadly conserved among yeasts and which were more species specific. Proteins were divided into several groups according to their role and localization. As expected, many Saccharomyces cerevisiae proteins involved in protein glycosylation, glycosylphosphatidylinositol (GPI) synthesis and the synthesis of wall polysaccharides had orthologues in most other yeasts. Similarly, a group of GPI anchored proteins involved in cell wall biosynthesis (Gas proteins and Dfg5p/Dcw1p) and other non-GPI anchored cell wall proteins involved in the wall synthesis and remodeling were highly conserved. However, GPI anchored proteins involved in flocculation, aggregation, cell separation, and those of still unknown functions were not highly conserved. The proteins localized in the cell walls of various yeast species were also analyzed by protein biotinylation and blotting. Pronounced differences were found both in the patterns, as well as in the overall amounts of different groups of proteins. The amount of GPI-anchored proteins correlated with the mannan to glucan ratio of the wall. Changes of the wall proteome upon temperature shift to 42 °C were detected.

Evolutionary Overview of Molecular Interactions and Enzymatic Activities in the Yeast Cell Walls.

Fungal cell walls are composed of a polysaccharide network that serves as a scaffold in which different glycoproteins are embedded. Investigation of fungal cell walls, besides simple identification and characterization of the main cell wall building blocks, covers the pathways and regulations of synthesis of each individual component of the wall and biochemical reactions by which they are cross-linked and remodeled in response to different growth phase and environmental signals. In this review, a survey of composition and organization of so far identified and characterized cell wall components of different yeast genera including Saccharomyces, Candida, Kluyveromyces, Yarrowia, and Schizosaccharomyces are presented with the focus on their cell wall proteomes.

Investigator Argus X-12 study on the population of northern Croatia.

X chromosome STR typing has emerged recently as a powerful tool, complementary to autosomal STR typing, in solving complex forensic and missing person cases. Investigator® Argus X-12 is a commercial product that allows co-amplification of 12 X chromosomal markers belonging to four linkage groups (LGs). In this study, we analyzed by capillary electrophoresis blood samples from 100 females and 102 males from a population of northern Croatia. Statistical analysis included calculation of allele and haplotype frequencies, as well as forensic parameters. The most informative marker for the northern Croatia population was DXS10135 with PIC=0.9211 and a total of 27 alleles. The least polymorphic marker was DXS8378 with 6 alleles. The proportion of observed haplotypes from the number of possible haplotypes varied from 2.74-8.57% across all LGs, with LG1 being the most informative. Of the 11 tested world populations compared to the population of northern Croatia, significant differences in genetic distance (FST) were found for Greenlandic and all non-European populations. We found that all tested markers are in HWE and can thus be used for match probability calculation. Because of high combined power of discrimination in both men and women, Investigator® Argus X-12 is applicable for the northern Croatia population in routine forensic casework.

Analysis of 12 X-chromosomal markers in the population of central Croatia.

Investigator® Argus X-12 Kit is a commercially available set that allows simultaneous PCR amplification of 12 X-STR markers belonging to four linkage groups (LG). To assess the forensic efficiency of these markers for the population of central Croatia and consequent applicability in routine forensic casework, DNA from 200 blood samples of unrelated donors (100 female and 100 male) was amplified by Investigator® Argus X-12 Kit and analyzed by capillary electrophoresis. Statistical computations based on allele and haplotype frequencies for LG1 - LG4 were performed using Arlequin 3.5 software and on-line tool available at ChrX-STR.org. In female samples, all X-STR markers were in Hardy-Weinberg equilibrium (HWE). The most informative marker for central Croatia population was DXS10135 with polymorphism information content (PIC) 0.9296. The least polymorphic locus was DXS8378 (PIC=0.6363). Power of discrimination (PD) varied from 0.6968 to 0.9336 in male and from 0.8476 to 0.9916 in female samples. Combined PD exceeded 0.999999999 in both men and women. In male samples, linkage disequilibrium (LD) test revealed significant association (P=0.0000) of one marker pair in LG4 and two marker pairs in LG3. Portion of observed haplotypes in the number of possible haplotypes varied from 2.86% to 7.47% across all LGs. LG1 was the most informative with haplotype diversity (H) 0.9972. High PD of all analyzed markers exhibited for central Croatia population confirms suitability of Investigator® Argus X-12 for forensic pertinence. Moreover, results of this study will be included in establishing a national reference X-STR database based on 12 X-STR loci, which is necessary for the correct interpretation of the forensic casework results.