Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.     

Topologies of a substrate protein bound to the chaperonin GroEL

The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.     

Growth factors improve gene expression after lentiviral transduction in human adult and fetal hepatocytes

BACKGROUND: Lentiviral vectors may be vectors of choice for transducing liver cells; they mediate integration in quiescent cells and offer potential for long-term expression. In adult liver, hepatocytes are generally mitotically quiescent. There has been controversy as to the necessity for lentiviral vector target cells to be in the cell cycle; currently, there is consensus that effective transduction can be achieved in quiescent hepatocytes, by using virus at high titre. However, transduction approaches which reduce the multiplicities of infection (MOIs) required provide potential benefit of cost and safety for therapeutic use. METHODS: We used two late-generation HIV-based lentiviral vector systems (pHR-SIN-cppT SGW and pRRLSIN.cPPT.PGK.WPRE) encoding LacZ/GFP reporter genes to transduce adult and fetal human hepatocytes in vitro + /- growth factors, hepatocyte growth factor (HGF) and epidermal growth factor (EGF). Green fluorescent protein (GFP) expression was observed microscopically, and quantified by fluorescence spectrometry for protein expression, fluorescence-activated cell sorting (FACS) analysis to identify the proportion of cells expressing GFP, and real-time quantitative polymerase chain reaction (PCR) for number of integrations. RESULTS: Gene expression following lentiviral transduction of human liver cells in vitro was markedly enhanced by the growth factors HGF and EGF. In adult cells growth factors led to a greater proportion of cells expressing more GFP per cell, from more integration events. In human fetal cells, the proportion of transduced hepatocytes remained identical, but cells expressed more GFP protein. CONCLUSIONS: This has implications for the design of regimes for liver cell gene therapy, allowing marked reduction of MOIs, and reducing both cost and risk of viral-mediated toxicity.     

Melanosome maturation defect in Rab38-deficient retinal pigment epithelium results in instability of immature melanosomes during transient melanogenesis

Pathways of melanosome biogenesis in retinal pigment epithelial (RPE) cells have received less attention than those of skin melanocytes. Although the bulk of melanin synthesis in RPE cells occurs embryonically, it is not clear whether adult RPE cells continue to produce melanosomes. Here, we show that progression from pmel17-positive premelanosomes to tyrosinase-positive mature melanosomes in the RPE is largely complete before birth. Loss of functional Rab38 in the "chocolate" (cht) mouse causes dramatically reduced numbers of melanosomes in adult RPE, in contrast to the mild phenotype previously shown in skin melanocytes. Choroidal melanocytes in cht mice also have reduced melanosome numbers, but a continuing low level of melanosome biogenesis gradually overcomes the defect, unlike in the RPE. Partial compensation by Rab32 that occurs in skin melanocytes is less effective in the RPE, presumably because of the short time window for melanosome biogenesis. In cht RPE, premelanosomes form but delivery of tyrosinase is impaired. Premelanosomes that fail to deposit melanin are unstable in both cht and tyrosinase-deficient RPE. Together with the high levels of cathepsin D in immature melanosomes of the RPE, our results suggest that melanin deposition may protect the maturing melanosome from the activity of lumenal acid hydrolases.     

Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability

Most sporadic colorectal tumors carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein is involved in many processes that govern gut tissue. In addition to its involvement in the regulation of beta-catenin, APC is a cytoskeletal regulator with direct and indirect effects on microtubules. Cancer-related truncation mutations lack direct and indirect binding sites for microtubules in APC, suggesting that loss of this function contributes to defects in APC-mutant cells. In this study, we show that loss of APC results in disappearance of cellular protrusions and decreased cell migration. These changes are accompanied by a decrease in overall microtubule stability and also by a decrease in posttranslationally modified microtubules in the cell periphery particularly the migrating edge. Consistent with the ability of APC to affect cell shape, the overexpression of APC in cells can induce cellular protrusions. These data demonstrate that cell migration and microtubule stability are linked to APC status, thereby revealing a weakness in APC-deficient cells with potential therapeutic implications.     

Repression of alpha-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract

Polypyrimidine tract binding protein (PTB) acts as a regulatory repressor of a large number of alternatively spliced exons, often requiring multiple binding sites in order to repress splicing. In one case, cooperative binding of PTB has been shown to accompany repression. The SM exon of the alpha-actinin pre-mRNA is also repressed by PTB, leading to inclusion of the alternative upstream NM exon. The SM exon has a distant branch point located 386 nt upstream of the exon with an adjacent 26 nucleotide pyrimidine tract. Here we have analyzed PTB binding to the NM and SM exon region of the alpha-actinin pre-mRNA. We find that three regions of the intron bind PTB, including the 3' end of the polypyrimidine tract (PPT) and two additional regions between the PPT and the SM exon. The downstream PTB binding sites are essential for full repression and promote binding of PTB to the PPT with a consequent reduction in U2AF(65) binding. Our results are consistent with a repressive mechanism in which cooperative binding of PTB to the PPT competes with binding of U2AF(65), thereby specifically blocking splicing of the SM exon.     

Differential expression of the keratan sulphate proteoglycan,keratocan, during chick corneal embryogenesis

Keratan sulphate (KS) proteoglycans (PGs) are key molecules in the connective tissue matrix of the cornea of the eye, where they are believed to have functional roles in tissue organisation and transparency. Keratocan, is one of the three KS PGs expressed in cornea, and is the only one that is primarily cornea-specific. Work with the developing chick has shown that mRNA for keratocan is present in early corneal embryogenesis, but there is no evidence of protein synthesis and matrix deposition. Here, we investigate the tissue distribution of keratocan in the developing chick cornea as it becomes compacted and transparent in the later stages of development. Indirect immunofluorescence using a new monoclonal antibody (KER-1) which recognises a protein epitope on the keratocan core protein demonstrated that keratocan was present at all stages investigated (E10–E18), with distinct differences in localisation and organisation observed between early and later stages. Until E13, keratocan appeared both cell-associated and in the stromal extracellular matrix, and was particularly concentrated in superficial tissue regions. By E14 when the cornea begins to become transparent, keratocan was located in elongate arrays, presumably associated along collagen fibrils in the stroma. This fibrillar label was still concentrated in the anterior stroma, and persisted through E15–E18. Presumptive Bowman’s layer was evident as an unlabelled subepithelial zone at all stages. Thus, in embryonic chick cornea, keratocan, in common with sulphated KS chains in the E12–E14 developmental period, exhibits a preferential distribution in the anterior stroma. It undergoes a striking reorganisation of structure and distribution consistent with a role in relation to stromal compaction and corneal transparency. E. Claire Gealy and Briedgeen C. Kerr were joint first authors.     

Locational distribution of gene functional classes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

We are interested in understanding the locational distribution of genes and their functions in genomes, as this distribution has both functional and evolutionary significance. Gene locational distribution is known to be affected by various evolutionary processes, with tandem duplication thought to be the main process producing clustering of homologous sequences. Recent research has found clustering of protein structural families in the human genome, even when genes identified as tandem duplicates have been removed from the data. However, this previous research was hindered as they were unable to analyse small sample sizes. This is a challenge for bioinformatics as more specific functional classes have fewer examples and conventional statistical analyses of these small data sets often produces unsatisfactory results.     

Annexins and endocytosis

Annexins are calcium- and phospholipid-binding proteins that have been proposed to have multiple roles in membrane traffic. Historically, this has been based on the in vitro properties of annexins and their localization to specific membrane compartments. However, recent functional evidence supports a role for annexins in specific membrane traffic steps, although the requirement for annexins may be highly dependent on the cellular context. Here we review the roles of annexins in traffic within the endocytic pathway, focusing on clathrin-dependent internalization from the plasma membrane, multivesicular endosome/body (MVB) biogenesis and MVB-lysosome fusion.     

Determinants of infant mortality and representation in bioarchaeological samples: A review

In bioarchaeological contexts, a complex relationship exists between infant representation in the age-at-death distribution, gestational and young child mortality rates, and the total fertility rate. The representation of infants in a skeletal sample may be influenced by a range of social, biological, and archaeological factors. To better understand the interactions between representation, fertility, and mortality, this study evaluates the relationship between infant-juvenile age-at-death proportions, fertility rates, and a range of gestational and early childhood mortality measures. The statistical component of this study found the correlation between fertility rates and infant-juvenile proportions was stronger than with any mortality rate variable of interest. This suggests that the proportion of infants in a mortuary sample is a stronger indicator of fertility than it is of infant-juvenile mortality. Social, biological, and archaeological variables potentially influencing infant representation in skeletal samples are discussed and a strongly contextualized and holistic approach to infant and juvenile mortality is recommended.