Highly phosphorylated bacterial proteins

We show in Gram-negative and Gram-positive bacteria the appearance of highly acidic proteins, which are highly phosphorylated. This group of proteins includes many cellular proteins, such as chaperones, biosynthetic, and metabolic enzymes. These proteins accumulate under stress conditions or under conditions, which overload the proteolytic system. Pulse chase experiments using radioactive phosphate indicate that the phosphorylated proteins have a short half-life, suggesting that they could be degradation intermediates. Moreover, results from in vitro experiments in Escherichia coli indicated that ribosomal proteins become susceptible to proteolysis after polyphosphorylation. Therefore, it is possible that the highly phosphorylated proteins represent a group of proteins tagged for degradation by phosphorylation. Such a tagging process may be involved in a general bacterial degradation pathway.     

Identification and characteristics of a novel E1 like gene nUBE1L in human testis

A gene, presumably involved in spermatogenesis, was identified and characterized by using cDNA microarray. Hybridization intensity was 2.13 fold higher in adult testis than that in fetal testis.The full length of this gene was 4288bp and it encoded a 578 amino acid protein. Conserved structure and amino acid sequence analysis revealed that the protein contained 1 Thif-domain, 2 UBACT-domains,and a functional active site cysteine lay upstream of UBACT domain, all of them also existed in ubiquitin-activating enzyme E1 and E1 like proteins. So we named this gene as a novel ubiquitin-activating enzyme E1 like gene (nUBE1L). Expression profile showed that nUBE1L was predominantly expressed in testis.Comparison of the expression of nUBE1L in different developmental stages of testis indicated that it was highly expressed in adult testis. In conclusion, nUBE1L is a novel human E1 like gene highly expressed inadult testis, which plays key role in ubiquitin system, and accordingly influences spermatogenesis and male fertility.     

Protein aggregation in Escherichia coli: role of proteases

Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases.     

The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases

The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.     

The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromosomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is approximately 45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, approximately 190 kDa; human, approximately 200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions. In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.     

A novel colonic repressor element regulates intestinal gene expression by interacting with Cux/CDP

Intestinal gene regulation involves mechanisms that direct temporal expression along the vertical and horizontal axes of the alimentary tract. Sucrase-isomaltase (SI), the product of an enterocyte-specific gene, exhibits a complex pattern of expression. Generation of transgenic mice with a mutated SI transgene showed involvement of an overlapping CDP (CCAAT displacement protein)-GATA element in colonic repression of SI throughout postnatal intestinal development. We define this element as CRESIP (colon-repressive element of the SI promoter). Cux/CDP interacts with SI and represses SI promoter activity in a CRESIP-dependent manner. Cux/CDP homozygous mutant mice displayed increased expression of SI mRNA during early postnatal development. Our results demonstrate that an intestinal gene can be repressed in the distal gut and identify Cux/CDP as a regulator of this repression during development.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.     

Premature termination of telomeric extension-PCR for detection of telomerase activity

In this article, we report a simple, rapid, and efficient method to detect telomerase activity: the premature termination of telomeric extension-PCR (PTEP). Similar to the telomeric repeat amplification protocol (TRAP), this method is based on PCR amplification following the in vitro telomerase reaction, while the in vitro telomerase reaction here is prematurely, rather than randomly, terminated. Apart from this, the telomeric extension products are used as initial primers, instead of as templates, to trigger the amplification with a specially constructed plasmid DNA as the template that cannot be directly amplified with the telomerase primer. The end product is a specific 159-bp DNA fragment that reflects telomerase activity. Because its product can be clearly identified with routine agarose gel electrophoresis and ethidium bromide staining, PTEP allows even lesser-equipped laboratories to easily detect telomerase activity.