IN02, A Positive Regulator of Lipid Biosynthesis, Is Essential for the Formation of Inducible Membranes in Yeast

Expression of the 180-kDa canine ribosome receptor in Saccharomyces cerevisiae leads to the accumulation of ER-like membranes. Gene expression patterns in strains expressing various forms of p180, each of which gives rise to unique membrane morphologies, were surveyed by microarray analysis. Several genes whose products regulate phospholipid biosynthesis were determined by Northern blotting to be differentially expressed in all strains that undergo membrane proliferation. Of these, the INO2 gene product was found to be essential for formation of p180-inducible membranes. Expression of p180 in ino2Delta cells failed to give rise to the p180-induced membrane proliferation seen in wild-type cells, whereas p180 expression in ino4Delta cells gave rise to membranes indistinguishable from wild type. Thus, Ino2p is required for the formation of p180-induced membranes and, in this case, appears to be functional in the absence of its putative binding partner, Ino4p.     

Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [(32)P]phosphoric acid to reveal a P3 intermediate. The (32)P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. (32)P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein.     

5 alpha-reductase-catalyzed conversion of testosterone to dihydrotestosterone is increased in prostatic adenocarcinoma cells: suppression by 15-lipoxygenase metabolites of gamma-linolenic and eicosapentaenoic acids

Although the androgens, testosterone (T) and its highly active metabolite dihydrotestosterone (DHT) play a role in the development and progression of prostate cancer, the mechanism(s) are unclear. Furthermore, 5 alpha-reductase which catalyze the conversion of T to DHT, has been a target of manipulation in the treatment of prostatic cancer, hence synthetic 5 alpha-reductase activity inhibitors have shown therapeutic promise. To demonstrate that nutrients derived from dietary sources can exert similar therapeutic promise, this study was designed using benign hyperplastic cells (BHC) and malignant tumorigenic cells (MTC) derived from Lobund-Wistar (L-W) rat model of prostatic adenocarcinoma to test the effects of gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and their 15-lipoxygenase metabolites on cellular 5 alpha-reductase activity. Our data revealed: (i) that incubation of MTC with [3H]-T resulted in marked conversion to [3H]-DHT when compared to similar incubation with BHC; (ii) that DHT-enhanced activity of 5 alpha-reductase was inhibited 80% by 15S-hydroxyeicosatrienoic acid, the 15-lipoxygenase metabolite of GLA, when compared to 55% by 15S-hydroxyeicosapentaenoic acid, the 15-lipoxygenase metabolite of EPA; and (iii) that their precursor fatty acids, respectively, exerted moderate inhibition. Taken together, the study underscores the biological importance of 15-lipoxygenase metabolites of polyunsaturated fatty acids (PUFAs) in androgen metabolism.     

Parasitic helminth infections in humans modulate Trefoil Factor levels in a manner dependent on the species of parasite and age of the host

Helminth infections, including hookworms and Schistosomes, can cause severe disability and death. Infection management and control would benefit from identification of biomarkers for early detection and prognosis. While animal models suggest that Trefoil Factor Family proteins (TFF2 and TFF3) and interleukin-33 (IL-33) -driven type 2 immune responses are critical mediators of tissue repair and worm clearance in the context of hookworm infection, very little is known about how they are modulated in the context of human helminth infection. We measured TFF2, TFF3, and IL-33 levels in serum from patients in Brazil infected with Hookworm and/or Schistosomes, and compared them to endemic and non-endemic controls. TFF2 was specifically elevated by Hookworm infection in females, not Schistosoma or co-infection. This elevation was correlated with age, but not worm burden. TFF3 was elevated by Schistosoma infection and found to be generally higher in females. IL-33 was not significantly altered by infection. To determine if this might apply more broadly to other species or regions, we measured TFFs and cytokine levels (IFNγ, TNFα, IL-33, IL-13, IL-1β, IL-17A, IL-22, and IL-10) in both the serum and urine of Nigerian school children infected with S. haematobium. We found that serum levels of TFF2 and 3 were reduced by infection, likely in an age dependent manner. In the serum, only IL-10 and IL-13 were significantly increased, while in urine IFN-γ, TNF-α, IL-13, IL-1β, IL-22, and IL-10 were significantly increased in by infection. Taken together, these data support a role for TFF proteins in human helminth infection.     

The C‐degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes

Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino‐ or carboxyl‐termini are eliminated by the N‐ or C‐degron pathways, respectively. We aimed to elucidate the function of C‐degron pathways and to unveil how normal proteomes are exempt from C‐degron pathway‐mediated destruction. Our data reveal that C‐degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C‐degron and N‐degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C‐degron pathways, but not of defective proteins, suggesting proteolysis‐based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.     

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

1. Download : Download high-res image (281KB)
2. Download : Download full-size image

     

Effects of randomizing the Sup35NM prion domain sequence on formation of amyloid fibrils in vitro

The mechanism by which proteins aggregate and form amyloid fibrils is still elusive. In order to preclude interference by cellular factors and to clarify the role of the primary sequence of Sup35p prion domain in formation of amyloid fibrils, we generated five Sup35NM variants by randomizing amino acid sequences in PrDs without altering the amino acid composition and analyzed the in vitro process of amyloid fibril formation. The results showed that each of the five Sup35NM variants polymerized into amyloid fibrils in vitro under native conditions. Furthermore, the Sup35NM variants showed differences in their aggregation time courses. These findings indicate that specific amino acid sequence features in PrD can modify the rate of conversion of Sup35p into amyloid fibrils in vitro.     

白喉毒素类免疫毒素研究进展

白喉毒素类免疫毒素是将缺失天然受体结合活性的白喉毒素片段或突变体与抗体或细胞因子偶联而得到的一类新型导向药物,它可特异性识别并结合靶细胞,通过发挥其ADP核糖基化活性而抑制细胞蛋白合成,引发细胞凋亡。由于白喉毒素类免疫毒素能高效、特异地杀伤特定靶细胞,而使其在肿瘤等疾病的药物开发中暂露头角。综述了基于白喉毒素的免疫毒素的研制现状与应用前景 。     

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.