Biochemistry and Cell Biology

Đã tiến hành nghiên cứu sự tương tác của một số peptide kháng khuẩn cationic giàu tryptophan (Trp) với màng. Những peptide này bao gồm tritrpticin, indolicidin, lactoferricin B (Lfcin B), và một đoạn ngắn hơn của lactoferricin (LfcinB_4–9). Môi trường trung bình của các gốc Trp của những peptide này được đánh giá từ tính chất phát huỳnh quang của chúng, cả độ dài sóng phát xạ cực đại lẫn hiệu ứng biên đỏ. Sự xâm nhập của các peptide vào các vesicle có thành phần khác nhau được kiểm tra thông qua sự dập tắt huỳnh quang Trp, với cả acrylamide hòa tan và các phospholipid gắn mác nitroxide, cũng như bằng cách sửa đổi hóa học các gốc Trp với N-bromosuccinimide. Kết quả thu được phù hợp với vị trí của các chuỗi bên Trp chủ yếu gần giao diện màng-nước. Mức độ chôn vùi của các chuỗi bên Trp có vẻ lớn hơn trong các vesicle chứa phospholipid với nhóm đầu phosphatidylglycerol âm. Sự rò rỉ của các thành phần nước trong liposome cũng được đo lường bằng cách sử dụng khảo sát bằng axit 8-aminonaphthalene-1,3,6-trisulfonic – p-xylene-bis-pyridinium bromide. Tritrpticin, thể hiện sự dịch chuyển biên đỏ lớn nhất, cũng hiển thị mức rò rỉ lớn nhất từ liposome. Tổng hợp lại, kết quả minh họa rằng các peptide kháng khuẩn giàu Trp cationic có xu hướng phá vỡ các túi đơn lớn mang điện tích âm tổng hợp sau khi chúng xâm nhập vào vùng giao diện của lớp kép phospholipid.

This paper reviews our current knowledge of the structure and function of the iron-binding protein lactoferrin. In particular, it attempts to relate the various proposed physiological functions of lactoferrin to its most characteristic biochemical properties, i.e. its ability to bind iron and its highly basic nature. The extent to which various physiological functions can be considered as definitely established is critically reviewed, and suggestions for future research are proposed.Key words: lactoferrin, iron, nutrition, immunology, infection, inflammation.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 µmol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 µmol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (≤100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.Key words: arginine kinase, guanidine-denatured, refolding, detergent, aggregation.

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8–1.0 mol/L and 0.3–0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8–1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3–0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.Key words: arginine kinase, guanidine-denatured, refolding, intermediate, molten globule state.

5-aminolevulinate synthase (ALAS; E.C. 2.3.1.37) catalyzes the first and rate-limiting step of heme synthesis within the mitochondria. Two isozymes of ALAS, encoded by separate genes, exist. ALAS1 is ubiquitously expressed and provides heme for cytochromes and other hemoproteins. ALAS2 is expressed exclusively in erythroid cells and synthesizes heme specifically for haemoglobin. A database search for proteins potentially regulated by oxygen tension revealed that ALAS2 contained a sequence of amino acids (LXXLAP where L is leucine, X is any amino acid, A is alanine, and P is proline) not occurring in ALAS1, which may be hydroxylated under normoxic conditions (21% O₂) and target the enzyme for ubiquitination and degradation by the proteasome. We examined protein turnover of ALAS2 in the presence of cycloheximide in K562 cells. Normoxic ALAS2 had a turnover time of approximately 36 h. Hypoxia (1% O₂) and inhibition of the proteasome increased both the stability and the specific activity of ALAS2 (greater than 2- and 7-fold, respectively, over 72 h of treatment). Mutation of a key proline within the LXXLAP sequence of ALAS2 also stabilized the protein beyond 36 h under normoxic conditions. The von Hippel-Lindau (vHL) protein was immunoprecipitated with FLAG epitope-tagged ALAS2 produced in normoxic cells but not in hypoxic cells, suggesting that the ALAS2 is hydroxylated under normoxic conditions and targeted for ubiquitination by the E3 ubiquitin ligase system. ALAS2 could also be ubiquitinated under normoxia using an in vitro ubiquitination assay. The present study provides evidence that ALAS2 is broken down under normoxic conditions by the proteasome and that the prolyl-4-hydroxylase/vHL E3 ubiquitin ligase pathway may be involved.Key words: erythroid-specific 5-aminolevulinate synthase, hypoxia, hydroxylation, prolyl-4-hydroxylases, E3 ubiquitin ligases, von Hippel-Lindau protein, proteasome.

The natural resistance-associated macrophage protein (Nramp) homologs form a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions (Me²⁺, including Mn²⁺, Fe²⁺, Co²⁺, and Cd²⁺). The Nramp, or solute carrier 11 (SLC11), family is conserved in eukaryotes and bacteria. Humans and rodents express 2 parologous genes that are associated with iron disorders and immune diseases. The NRAMP1 (SLC11A1) protein is specific to professional phagocytes and extrudes Me²⁺from the phagosome to defend against ingested microbes; polymorphisms in the NRAMP1 gene are associated with various immune diseases. Several isoforms of NRAMP2 (SLC11A2, DMT1, DCT1) are expressed ubiquitously in recycling endosomes or specifically at the apical membrane of epithelial cells in intestine and kidneys, and can contribute to iron overload, whereas mutations impairing NRAMP2 function cause a form of congenital microcytic hypochromic anemia. Structure–function studies, using various experimental models, and mutagenesis approaches have begun to reveal the overall transmembrane organization of Nramp, some of the transmembrane segments (TMS) that are functionally important, and an unusual mechanism coupling Me²⁺and proton H⁺transport. The approaches used include functional complementation of yeast knockout strains, electrophysiology analyses in Xenopus oocytes, and transport assays that use mammalian and bacterial cells and direct and indirect measurements of SLC11 transporter properties. These complementary studies enabled the identification of TMS1and 6 as crucial structural segments for Me²⁺and H⁺symport, and will help develop a deeper understanding of the Nramp transport mechanism and its contribution to Me²⁺homeostasis in human health and diseases.

The structure, dynamics, and DNA-binding characteristics of wild-type and cross-linked Cro repressors are compared by using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The Cro repressor is a small dimeric DNA-binding protein from bacteriophage λ. Replacement of valine-55 by cysteine in the dimer interaction region of each monomer subunit results in the spontaneous formation of a disulfide cross-link between the subunits. Two-dimensional nuclear Overhauser effect spectroscopy and CD data show the variant has nearly the same conformation as the wild-type protein. However, by monitoring the CD band at 222 nm, the cross-linked protein is shown to have a heat-denaturation midpoint temperature of 67 °C, whereas the wild-type protein has a melting temperature of about 47 °C. Using ¹H-NMR to follow the denaturation by heat, the same melting temperature is observed for wild-type Cro (47 °C), but a much lower melting temperature is seen for V55C Cro (58 °C). This suggests that between 58 and 67 °C the cross-linked protein exists in a molten globule state with the α-helices mainly intact, but without the interaction of chemical groups that cause spectral dispersion. Binding parameters for interaction of the proteins with DNA were obtained by observing the NMR spectrum for the imino protons of a 10 base-pair half-operator DNA and titrating in protein. The cross-linked protein binds DNA (K_d = 160 μM) about eight times more weakly than the wild-type protein (K_d = 19 μM). Adjustments in protein structure, necessary to form a tight protein-DNA complex, appear to be hindered by a loss in protein flexibility caused by the intersubunit cross-link.Key words: nuclear magnetic resonance, circular dichroism, Cro repressor, protein engineering, disulfide bond.

The incorporation of [methyl-¹⁴C]CDP-choline into phosphatidylcholine was measured in HeLa cells permeabilized with 0.125 mg digitonin/mL. The rate of phosphatidylcholine formation was influenced by the concentration of CDP-choline in the medium. The CDP-choline: 1,2-diacylglycerol cholinephosphotransferase in permeabilized cells showed a K_m of 88 μM for CDP-choline. A similar K_m value of 104 μM was found for cholinephosphotransferase in microsomes isolated from HeLa cells when assayed in the presence of 2.4 mM dioleoylglycerol. In the absence of added diacylglycerol, the K_m for CDP-choline for the microsomal cholinephosphotransferase was only 38 μM. The incorporation of [methyl-¹⁴C]CDP-choline into phosphatidylcholine was stimulated by the supply of diacylglycerol in both HeLa cells and isolated microsomes. A 2.4 mM dioleoylglycerol suspension increased cholinephosphotransferase activity fourfold in microsomes. The digitonin-treated cells were impermeable to the dioleoylglycerol suspension. Incubation of permeabilized cells with 150 μM acyl-CoA and 0.8 mM glycero-3-phosphate tripled cellular diacylglycerol levels, causing a doubling in the rate of phosphatidylcholine synthesis. A similar incubation of microsomes with acyl-CoA stimulated phosphatidylcholine synthesis twofold. Furthermore, incubation of microsomes with [³H]diacylglycerol and [¹⁴C]CDP-choline showed that both of the substrates were incorporated into phosphatidylcholine at the same rate. This result suggests that the stimulatory effects on cholinephosphotransferase arise from increases in the availability of substrates rather than activation of the enzyme. These results suggest that both in the permeabilized cells and in isolated membranes, the biosynthesis of phosphatidylcholine can be limited by both CDP-choline and diacylglycerol.

Stimulation of the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway results in a multitude of events including expression of the immediate-early genes, c-fos and c-myc. Downstream targets of this stimulated pathway are the mitogen- and stress-activated protein kinases (MSK) 1 and 2, which are histone H3 kinases. In chromatin immunoprecipitation assays, it has been shown that the mitogen-induced phosphorylated H3 is associated with the immediate-early genes and that MSK1/2 activity and H3 phosphorylation have roles in chromatin remodeling and transcription of these genes. In oncogene-transformed fibroblasts in which the Ras-MAPK pathway is constitutively active, histone H1 and H3 phosphorylation is increased and the chromatin of these cells has a more relaxed structure than the parental cells. In this review we explore the deregulation of the Ras-MAPK pathway in cancer, with an emphasis on breast cancer. We discuss the features of MSK1 and 2 and the impact of a constitutively activated Ras-MAPK pathway on chromatin remodeling and gene expression.Key words: Ras, mitogen-activated protein kinase signal transduction pathway, histone H3 phosphorylation, MSK1, breast cancer.