Assignment of protein backbone resonances using connectivity, torsion angles and 13Cα chemical shifts

A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just (13)C(alpha) chemical shifts (delta((13)C(alpha))) and data restricting the phi and psi backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in delta((13)C(alpha)), phi, and psi space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and (13)C(alpha) chemical shift data are available.     

Backbone 1H, 15N, 13C and side chain 13Cβ NMR chemical shift assignment of murine interleukin-10

Almost complete assignment of backbone ¹H, ¹³C, ¹⁵N and side chain ¹³Cβ resonances for the immune-regulatory cytokine IL-10 is reported. The protein was overexpressed in Escherichia coli and was refolded from inclusion bodies. The point mutation C149Y was introduced to suppress incorrect disulfide bond formation and to improve protein refolding.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Clinicopathological Correlation and Prognostic Significance of Protein Kinase Cα Overexpression in Human Gastric Carcinoma

Objectives

This study investigated the PKCα protein expression in gastric carcinoma, and correlated it with clinicopathological parameters. The prognostic significance of PKCα protein expression in gastric carcinoma was analyzed.

Methods

Quantitative real-time PCR test was applied to compare the PKCα mRNA expression in tumorous and nontumorous tissues of gastric carcinoma in ten randomly selected cases. Then PKCα protein expression was evaluated in 215 cases of gastric carcinoma using immunohistochemical method. The immunoreactivity was scored semiquantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely PKCα overexpression group with score 2 or 3, and non-overexpression group with score 0 or 1. The PKCα protein expression was correlated with clinicopathological parameters. Survival analysis was performed to determine the prognostic significance of PKCα protein expression in patients with gastric carcinoma.

Results

PKCα mRNA expression was upregulated in all ten cases of gastric carcinoma via quantitative real-time PCR test. In immunohistochemical study, eighty-eight out of 215 cases (41%) of gastric carcinoma revealed PKCα protein overexpression, which was statistically correlated with age (P = 0.0073), histologic type (P<0.0001), tumor differentiation (P = 0.0110), depth of invasion (P = 0.0003), angiolymphatic invasion (P = 0.0373), pathologic stage (P = 0.0047), and distant metastasis (P = 0.0048). We found no significant difference in overall and disease free survival rates between PKCα overexpression and non-overexpression groups (P = 0.0680 and 0.0587). However, PKCα protein overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 0.632, P = 0.0415).

Conclusions

PKCα protein is upregulated in gastric carcinoma. PKCα protein expression is statistically correlated with age, histologic type, tumor differentiation, depth of invasion, angiolymphatic invasion, pathologic stage, and distant metastasis. The PKCα protein overexpression in patients with gastric carcinoma is a significant independent prognostic factor in multivariate Cox regression analysis.     

Functions of Aβ, sAPPα and sAPPβ : similarities and differences

Amyloid peptide (Aβ) is derived from the cleavage of amyloid precursor protein (APP), which also generates the soluble peptide APPβ (sAPPβ). An antagonist and major APP metabolic pathway involves cleavage by alpha secretase, which releases sAPPα. Although soluble Aβ oligomers are neurotoxic, Aβ monomers share similar properties with sAPPα. These include neurotrophic and neuroprotective effects, as well as stimulation of neural-progenitor proliferation. The properties of Aβ monomers and the neurotrophic capacity of sAPPβ to stimulate axonal outgrowth suggest that Aβ production is not deleterious per se. Consequently, therapeutic strategies for Alzheimer's disease that are targeted at Aβ-cleaving enzymes should modulate rather than inhibit Aβ generation. These strategies should focus on the factors that induce the conversion of Aβ monomers into toxic soluble oligomers. Another interesting therapeutic approach is to focus on the mechanisms of the different properties of sAPPα. Indeed, increasing sAPPα levels could shift proliferating cells towards tumorigenesis. In contrast to its neuroprotective effects, sAPPα is also able to activate microglia, leading to neurotoxicity. Understanding the mechanisms that underlie the different properties of sAPPα could therefore lead to the development of therapeutic strategies against Alzheimer's disease, which could be curative as well as preventive.     

Resonance assignment of nsp7α from arterivirus

The ¹H, ¹⁵N and ¹³C resonance assignment of nsp7α, a non-structural protein of unknown function from the equine arteritis virus, is reported.     

Identification of Zinc-ligated Cysteine Residues Based on 13Cα and 13Cβ Chemical Shift Data

Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure and/or play critical catalytic roles in the underlying biochemistry. An atom’s chemical shift is exquisitely sensitive to its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation. In this study, we demonstrate that overlapped ¹³Cβ chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion of the corresponding ¹³Cα chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using 287 ¹³Cα/¹³Cβ shift pairs from 79 proteins with known three-dimensional structures, including 86 ¹³Cα and¹³Cβ shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information, we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines, and Zn-ligated cysteines. Classifying cysteines/cystines with a statisical model incorporating all three phenomena resulted in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron–sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands. We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn ligands, we analyzed the Protein Data Bank and found that 78% of the Zn ligands are histidine and cysteine (with nearly identical frequencies), and 18% are acidic residues aspartate and glutamate. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Genomic organization of human complement protein C8α and further examination of its linkage to C8β

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the cytolytic C5b-9 complex on target membranes. It is composed of three nonidentical subunits (C8, C8, C8) encoded by separate genes. C8 and C8 are linked on chromosome 1p32, whereas C8 is located on 9q22.3-q32. In this study, overlapping genomic clones were isolated and used to decipher the organization of the human C8 gene. The gene contains at least 11 exons spanning 70kb of DNA. When compared to C6, C8 and C9, there is a remarkable similarity in genomic organization, consistent with amino acid sequence comparisons that suggest these proteins are ancestrally related. Regions of each protein that are structurally similar are encoded in exons of correspondingly similar lengths with highly conserved boundaries and phases. Availability of genomic sequence also facilitated a more detailed analysis of C8 and C8 linkage. Based on analysis of genomic digests with cDNA probes, the loci were previously reported to be physically linked (< 2.5kb) and in a 5- 3 orientation. In the present study, results obtained using exon-specific probes indicate the loci are not as closely linked as initially believed. Furthermore, they suggest that cDNA probes used earlier yielded misleading information because they encode exons that are distributed across large segments of genomic DNA.     

Lipopolysaccharide-induced Overproduction of Nitric Oxide and Overexpression of iNOS and Interleukin-1β Proteins in Zinc-deficient Rats

Zinc deficiency leads to decreased cellular immune responses. The overproduction of nitrogen species derived from inducible nitric oxide synthase (iNOS), its enzyme, and interleukine-1 beta (IL-1β), and inflammatory cytokine have been implicated in immune responses. The goal of this study was to investigate the effects of lipopolysaccharide (LPS)-induced changes in NO metabolites, iNOS, and IL-1β protein expression in the lungs of zinc-deficient rats. Male Sprague–Dawley rats (body weight, 100 g) were divided into two groups and were fed either a zinc-deficient diet (ZnD) or a zinc-containing diet (Cont). After 4 weeks on these diets, rats received a 10-mg/kg dose of LPS injected via the tail vein and were then maintained for an additional 72 h. To determine total NO concentrations in the blood, serum zinc concentration, iNOS protein expression, IL-1β, and iNOS immunohistochemistry, blood and lung samples were obtained at pre-LPS injection, 5, 24, and 72 h after injection. Total NO levels were significantly increased at 5, at 24, and at 72 h after LPS injection compared with pre-LPS injection level in ZnD group; significant changes in total NO levels was elevated at 5 h from at pre-LPS level but not significant changes from basal level at 24 and 72 h in the control group. Based on western blot analyses and immunohistochemistry, clear bands indicating iNOS and IL-1β protein expression and iNOS antibody-stained inflammatory cells were detected at 5 and 24 h in the ZnD group and 5 h in the Cont group, not observed at 24 and 72 h in the control group. These results suggest that zinc deficiency induces overexpression of iNOS and IL-1β proteins from inflammatory cells around the alveolar blood vessels, resulting in overproduction of total NO and persisted inflammatory response in the zinc-deficient rat lung. Taken together, overexpression of LPS-induced iNOS, overproduction of iNOS-derived NO, and overexpression of IL-1β may induce nitrosative and oxidative stresses in the lung, and these stresses may be involved low immunity of zinc deficiency states.     

IKKα and IKKβ Regulation of DNA Damage-Induced Cleavage of Huntingtin

Background

Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington''s disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IκB kinase (IKK), which can influence neurodegeneration in HD.

Methodology/Principal Findings

We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or γ-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80–90 kDa. This event requires IKKβ and is suppressed by IKKα. Elevated levels of IKKα, or inhibition of IKKβ expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKβ phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKβ expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKβ, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis.

Conclusions/Significance

These results indicate that DNA damage triggers cleavage of Htt and identify IKKβ as a prominent regulator. Moreover, IKKβ-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKβ may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders.     

Protein phosphatase type 2Cα and 2Cβ are involved in fatty acid-induced apoptosis of neuronal and endothelial cells

Unsaturated fatty acids with special structural features have been shown to activate serine/threonine protein phosphatase type 2C (PP2C) isoforms α and β at physiological Mg²⁺-concentrations in vitro. These compounds also induce apoptosis in neuronal and endothelial cells. In this study we further analysed this striking correlation and tried to elucidate whether or not there is a causative relationship between activation of PP2C and induction of apoptosis. We employed RNA interference to simultaneously knock down PP2Cα and PP2Cβ in SH-SY5Y cells or HUVECs, respectively. This downregulation was transient. Treatment of SH-SY5Y cells or HUVECs with oleic acid (18:1,cis-Δ⁹) caused apoptosis in a time- and concentration-dependent manner. In both cases, cells with reduced PP2C-levels were less susceptible to oleic acid-induced cell damage. In conclusion, our results demonstrate that PP2C activation by unsaturated fatty acids actually causes apoptosis in neuronal and endothelial cells.     

13Cα decoupling during direct observation of carbonyl resonances in solution NMR of isotopically enriched proteins

Direct detection of ¹³C can be advantageous when studying uniformly enriched proteins, in particular for paramagnetic proteins or when hydrogen exchange with solvent is fast. A scheme recently introduced for long-observation-window band-selective homonuclear decoupling in solid state NMR, LOW-BASHD (Struppe et al. in J Magn Reson 236:89–94, 2013) is shown to be effective for ¹³C^α decoupling during direct ¹³C′ observation in solution NMR experiments too. For this purpose, adjustment of the decoupling pulse parameters and delays is demonstrated to be important for increasing spectral resolution, to reduce three-spin effects, and to decrease the intensity of decoupling side-bands. LOW-BASHD then yields ¹³C′ line widths comparable to those obtained with the popular IPAP method, while enhancing sensitivity by ca 35 %. As a practical application of LOW-BASHD decoupling, requiring quantitative intensity measurement over a wide dynamic range, the impact of lipid binding on the ¹³C′-detected NCO spectrum of the intrinsically disordered protein α-synuclein is compared with that on the ¹H-detected ¹H–¹⁵N HSQC spectrum. Results confirm that synuclein’s “dark state” behavior is not caused by paramagnetic relaxation or rapid hydrogen exchange.     

The correlation between multidomain enzymes and multiple activation mechanisms--the case of phospholipase Cβ and its membrane interactions

Phospholipase Cβ2 (PLCβ2) is a large, multidomain enzyme that catalyzes the hydrolysis of the signaling lipid phosphoinositol 4,5 bisphosphate (PIP2) to promote mitogenic and proliferative changes in the cell. PLCβ2 is activated by Gα and Gβγ subunits of heterotrimeric G proteins, as well as small G proteins and specific peptides. Activation depends on the nature of the membrane surface. Recent crystal structures suggest one model of activation involving the movement of a small autoinhibitory loop upon membrane binding of the enzyme. Additionally, solution studies indicate multiple levels of activation that involve changes in the membrane orientation as well as interdomain movement. Here, we review the wealth of biochemical studies of PLCβ2-G protein activation and propose a comprehensive model that accounts for both the crystallographic and solution results.     

Cα and Cβ Carbon-13 Chemical Shifts in Proteins From an Empirical Database

We have constructed an extensive database of 13C Cα and Cβ chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical α- helix and β-sheet geometries for Cα, and of ca. 2 ppm for Cβ. The side-chain dihedral angle χ1 has an effect of up to 0.5 ppm on the Cα shift, particularly for amino acids with branched side-chains at Cβ. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main- chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different φ,ψ shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and χ1 angle, is 0.96 ppm for both Cα and Cβ. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.