Differential phosphopeptide expression in a benign breast tissue, and triple-negative primary and metastatic breast cancer tissues from the same African-American woman by LC-LTQ/FT-ICR mass spectrometry

African-American women have a higher risk for developing triple-negative breast cancer (TNBC). Lacking the expression of receptors for estrogen and progesterone, and without human epidermal growth factor 2 receptor gene amplification, TNBC is a very aggressive type of breast cancer with a high likelihood of metastasis and recurrence. Specific therapeutic targets for this aggressive disease remain to be identified. Phosphorylation, a post-translational modification that adds one or more phosphate groups to a protein, plays a key role in the activation and deactivation of a protein’s cellular function. Here, we report the first systematic phosphoproteomic analysis of a benign breast tissue, a primary breast cancer tissue, and a metastatic breast cancer tissue from the same African-American woman. Differential phosphoprotein levels were measured with reversed-phase nano-liquid chromatography coupled to a hybrid linear quadrupole ion trap/Fourier transform ion cyclotron resonance mass spectrometer (LC-LTQ/FT-ICR MS). Five proteins were found to be highly phosphorylated in the metastatic site whereas six proteins were highly phosphorylated in the cancer site of the TNBC patient. Identified phosphoproteins are known to be involved in breast cancer signal transduction pathways and these results may identify new diagnostic and therapeutic targets for TNBC.     

Intramembrane Water Associated with TOAC Spin-Labeled Alamethicin: Electron Spin-Echo Envelope Modulation by D2O

Alamethicin is a 20-residue, hydrophobic, helical peptide, which forms voltage-sensitive ion channels in lipid membranes. The helicogenic, nitroxyl amino acid TOAC was substituted isosterically for Aib at residue positions 1, 8, or 16 in a F50/5 alamethicin analog to enable EPR studies. Electron spin-echo envelope modulation (ESEEM) spectroscopy was used to investigate the water exposure of TOAC-alamethicin introduced into membranes of saturated or unsaturated diacyl phosphatidylcholines that were dispersed in D₂O. Echo-detected EPR spectra were used to assess the degree of assembly of the peptide in the membrane, via the instantaneous diffusion from intermolecular spin-spin interactions. The profile of residue exposure to water differs between membranes of saturated and unsaturated lipids. In monounsaturated dioleoyl phosphatidylcholine, D₂O-ESEEM intensities decrease from TOAC¹ to TOAC⁸ and TOAC¹⁶ but not uniformly. This is consistent with a transmembrane orientation for the protoassembled state, in which TOAC¹⁶ is located in the bilayer leaflet opposite to that of TOAC¹ and TOAC⁸. Relative to the monomer in fluid bilayers, assembled alamethicin is disposed asymmetrically about the bilayer midplane. In saturated dimyristoyl phosphatidylcholine, the D₂O-ESEEM intensity is greatest for TOAC⁸, indicating a more superficial location for alamethicin, which correlates with the difference in orientation between gel- and fluid-phase membranes found by conventional EPR of TOAC-alamethicin in aligned phosphatidylcholine bilayers. Increasing alamethicin/lipid ratio in saturated phosphatidylcholine shifts the profile of water exposure toward that with unsaturated lipid, consistent with proposals of a critical concentration for switching between the two different membrane-associated states.     

14-3-3 proteins in neurodegeneration

Among the first reported functions of 14-3-3 proteins was the regulation of tyrosine hydroxylase (TH) activity suggesting a possible involvement of 14-3-3 proteins in Parkinson's disease. Since then the relevance of 14-3-3 proteins in the pathogenesis of chronic as well as acute neurodegenerative diseases, including Alzheimer's disease, polyglutamine diseases, amyotrophic lateral sclerosis and stroke has been recognized. The reported function of 14-3-3 proteins in this context are as diverse as the mechanism involved in neurodegeneration, reaching from basal cellular processes like apoptosis, over involvement in features common to many neurodegenerative diseases, like protein stabilization and aggregation, to very specific processes responsible for the selective vulnerability of cellular populations in single neurodegenerative diseases.Here, we review what is currently known of the function of 14-3-3 proteins in nervous tissue focussing on the properties of 14-3-3 proteins important in neurodegenerative disease pathogenesis.     

Proteomic analysis of B-cell malignancies

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.     

Deletions in the A(L) region of the h4xb plasma membrane Ca(2+) pump. High apparent affinity for Ca(2+) of a deletion mutant resembling the alternative spliced form h4zb

Mutants of the plasma membrane Ca(2+) pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (A(L) region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300-349 did not change the maximal Ca(2+) transport activity, but mutants with deletions involving residues 300-338 exhibited a higher apparent affinity for Ca(2+) than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339-349) had no observable functional consequences. The removal of either residues 300-314 or 313-338 resulted in a similar increase in the apparent Ca(2+) affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300-349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca(2+) of the human isoform 4 of the PMCA.     

Molecular basis of albinism in India: Evaluation of seven potential candidate genes and some new findings

Albinism represents a group of genetic disorders with a broad spectrum of hypopigmentary phenotypes dependent on the genetic background of the patients. Oculocutaneous albinism (OCA) patients have little or no pigment in their eyes, skin and hair, whereas ocular albinism (OA) primarily presents the ocular symptoms, and the skin and hair color may vary from near normal to very fair. Mutations in genes directly or indirectly regulating melanin production are responsible for different forms of albinism with overlapping clinical features. In this study, 27 albinistic individuals from 24 families were screened for causal variants by a PCR-sequencing based approach. TYR, OCA2, TYRP1, SLC45A2, SLC24A5, TYRP2 and SILV were selected as candidate genes. We identified 5 TYR and 3 OCA2 mutations, majority in homozygous state, in 8 unrelated patients including a case of autosomal recessive ocular albinism (AROA). A homozygous 4-nucleotide novel insertion in SLC24A5 was detected in a person showing with extreme cutaneous hypopigmentation. A potential causal variant was identified in the TYRP2 gene in a single patient. Haplotype analyses in the patients carrying homozygous mutations in the classical OCA genes suggested founder effect. This is the first report of an Indian AROA patient harboring a mutation in OCA2. Our results also reveal for the first time that mutations in SLC24A5 could contribute to extreme hypopigmentation in humans.     

The phylogeny of Ephemeroptera in Pterygota revealed by the mitochondrial genome of Siphluriscus chinensis (Hexapoda: Insecta)

The mayfly species Siphluriscus chinensis (Siphluriscidae) has valuable structures useful for phylogeny reconstruction, given its putative basal position within the Ephemeroptera. Here its nearly complete mitochondrial genome is sequenced. We built phylogenetic trees through multiple analytical strategies with some other insect mitogenomes. Structurally, the obtained mitochondrial genome of S. chinensis is 16,616 bp in length, ¹ containing 37 genes and an extra trnK-like (trnK2 (AAA)) gene. The 12 PCGs start with typical ATN codons, except the nad1 gene which starts with an unnormalized TTG. Like other known mayfly mitogenomes, the strand bias has negative AT-skew and negative GC-skew. Phylogenetically, our topologies suggest that Odonata is the basally diverged clade in Pterygota; Ephemeroptera is the sister group of the Neoptera; and S. chinensis is indeed the most basal mayfly branch.