Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif.

Import of proteins into peroxisomes depends on PEX5, PEX13 and PEX14. By combining biochemical methods and structural biology, we show that the C-terminal SH3 domain of PEX13 mediates intramolecular interactions with a proximal FxxxF motif. The SH3 domain also binds WxxxF peptide motifs in the import receptor PEX5, demonstrating evolutionary conservation of such interactions from yeast to human. Strikingly, intramolecular interaction of the PEX13 FxxxF motif regulates binding of PEX5 WxxxF/Y motifs to the PEX13 SH3 domain. Crystal structures reveal how FxxxF and WxxxF/Y motifs are recognized by a non-canonical surface on the SH3 domain. The PEX13 FxxxF motif also mediates binding to PEX14. Surprisingly, the potential PxxP binding surface of the SH3 domain does not recognize PEX14 PxxP motifs, distinct from its yeast ortholog. Our data show that the dynamic network of PEX13 interactions with PEX5 and PEX14, mediated by diaromatic peptide motifs, modulates peroxisomal matrix import.

Comparison of human PEX knockout cell lines suggests a dual role of PEX1 in peroxisome biogenesis.

For the biogenesis and maintenance of peroxisomes several proteins, called peroxins, are essential. Malfunctions of these proteins lead to severe diseases summarized as peroxisome biogenesis disorders. The different genetic background of patient-derived cell lines and the residual expression of mutated PEX genes impede analysis of the whole spectrum of cellular functions of affected peroxins. To overcome these difficulties, we have generated a selected PEX knockout resource of HEK T-REx293 cells using the CRISPR/Cas9 technique. Comparative analyses of whole cell lysates revealed PEX-KO specific alterations in the steady-state level of peroxins and variations in the import efficacy of matrix proteins with a Type 2 peroxisomal targeting signal. One of the observed differences concerned PEX1 as in the complete absence of the protein, the number of peroxisomal ghosts is significantly increased. Upon expression of PEX1, import competence and abundance of peroxisomes was adjusted to the level of normal HEK cells. In contrast, expression of an alternatively spliced PEX1 isoform lacking 321 amino acids of the N-terminal region failed to rescue the peroxisomal import defects but reduced the number of peroxisomal vesicles. All in all, the data suggest a novel 'moonlighting' function of human PEX1 in the regulation of pre-peroxisomal vesicles.

Screening Pregnant Women for Bacterial Vaginosis Using a Point-of-Care Test: A Prospective Validation Study.

Bacterial vaginosis in early pregnancy is associated with an increased risk of preterm birth. The introduction of a simple screen-and-treat program into antenatal care was shown to significantly reduce the rate of preterm birth. The gold standard for diagnosing bacterial vaginosis is Gram staining, which is, however, time-consuming and requires laboratory facilities. The objective of this prospective study was to validate a point-of-care sialidase activity detection test (OSOM® BVBLUE® Test) for asymptomatic pregnant women and evaluate its accuracy as a screening tool. We enrolled 200 pregnant participants, 100 with Gram staining-confirmed bacterial vaginosis and 100 healthy controls. Compared to Gram staining, the point-of-care test showed a sensitivity of 81%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 98.1%. In conclusion, we found that the OSOM® BVBLUE® Test was an accurate method for diagnosing bacterial vaginosis in asymptomatic pregnant women. This point-of-care test can therefore be considered a reliable and easy-to-use screening tool for bacterial vaginosis during pregnancy.

Validation of the SavvyCheck™ Vaginal Yeast Test for Screening Pregnant Women for Vulvovaginal Candidosis: A Prospective, Cross-Sectional Study.

Pregnant women have an increased risk of vulvovaginal candidosis. Recurrent candidosis is under debate as a contributor to preterm birth, and vertical transmission may cause diaper dermatitis and oral thrush in the newborn. Apart from cultural methods, the gold standard for diagnosing candidosis is Gram staining, which is time-consuming and requires laboratory facilities. The objective of this prospective study was to validate a point-of-care vaginal yeast detection assay (SavvyCheck™ Vaginal Yeast Test) and to evaluate it in asymptomatic pregnant women. We enrolled 200 participants, 100 of whom had vulvovaginal candidosis according to Gram stain (study group) and 100 were healthy pregnant controls (control group). Of these, 22 participants (11%) had invalid test results. The point-of-care test of the remaining 85 and 93 study participants in the study and control groups, respectively, showed a sensitivity of 94.1%, specificity of 98.9%, positive predictive value of 90.3%, and negative predictive value of 99.4% when compared with Gram stain. In conclusion, we found a high correlation between the SavvyCheck™ Vaginal Yeast Test and Gram-stained smears during pregnancy. This suggests a potential role of this point-of-care test as a screening tool for asymptomatic pregnant women in early gestation.

Novel Trypanocidal Inhibitors that Block Glycosome Biogenesis by Targeting PEX3-PEX19 Interaction.

Human pathogenic trypanosomatid parasites harbor a unique form of peroxisomes termed glycosomes that are essential for parasite viability. We and others previously identified and characterized the essential Trypanosoma brucei ortholog TbPEX3, which is the membrane-docking factor for the cytosolic receptor PEX19 bound to the glycosomal membrane proteins. Knockdown of TbPEX3 expression leads to mislocalization of glycosomal membrane and matrix proteins, and subsequent cell death. As an early step in glycosome biogenesis, the PEX3-PEX19 interaction is an attractive drug target. We established a high-throughput assay for TbPEX3-TbPEX19 interaction and screened a compound library for small-molecule inhibitors. Hits from the screen were further validated using an in vitro ELISA assay. We identified three compounds, which exhibit significant trypanocidal activity but show no apparent toxicity to human cells. Furthermore, we show that these compounds lead to mislocalization of glycosomal proteins, which is toxic to the trypanosomes. Moreover, NMR-based experiments indicate that the inhibitors bind to PEX3. The inhibitors interfering with glycosomal biogenesis by targeting the TbPEX3-TbPEX19 interaction serve as starting points for further optimization and anti-trypanosomal drug development.