Swiss Protein Research Articles

Analysis of Phylogenetics and Structural Relationships of the Cytochrome b Gene Protein in Mahseer Fish (Tor sp.)

Abstract The Cytochrome b gene from mitochondrial DNA encodes a protein that can be used as a genetic marker to identify species. This study aims to determine the bioinformatic characteristics of the gene encoding Cyt b isolated from various Mahseer species, Tor tambroides, Tor douronensis, Tor khudree, and Tor putitora. Mahseer Cyt b sequences can be used for detailed computational investigation of the properties of 3D protein models and phylogenetics. The data analysis technique uses nucleotides with a sequence length of 330 bp obtained from NCBI. The MEGA X program was used for phylogenetic analysis. 3D protein structure predictions were obtained through the Swiss model and protein homology program using PYMOL software. The results of phylogenetics show close kinship relationships with the closest genetic distance of 0.000, while the farthest distance is 0.068. The nucleotide composition contains Thymine (30.23%), Cytosine (25.81%), Arginine (28.05%), and Guanine (15.92%). 3D modeling of the Cyt b protein Mahseer referring to the species Neolissochilus hexagonolepis (McClelland, 1839). The 3D protein structure prediction was assessed via a Ramachandran plot of 99.07%. This study can be used as a reference to support sustainable conservation efforts by observing the populations and habitat conditions.

Synthesis, in silico screening, and biological evaluation of novel pyridine congeners as anti-epileptic agents targeting AMPA (α-amino-3-hydroxy-5-methylisoxazole) receptors.

The research involves the synthesis of a series of new pyridine analogs 5(i-x) and their evaluation for anti-epileptic potential using in silico and invivo models. Synthesis of the compounds was accomplished by using the Vilsmeier-Haack reaction principle. AutoDock 4.2 was used for their in silico screening against AMPA (-amino-3-hydroxy-5-methylisoxazole) receptor (PDB ID:3m3f). For invivo testing, the maximal electroshock seizure (MES) model was used. The physicochemical, pharmacokinetic, drug-like, and drug-score features of all synthesized compounds were assessed using the online Swiss ADME and Protein Plus software. The in silico results showed that all the synthesized compounds 5(i-x) had 1-3 interactions and affinities ranging from -6.5 to -8.0 kJ/mol with the targeted receptor compared to the binding affinities of the standard drug phenytoin and the original ligand of the target (P99), which were -7.6 and -6.8 kJ/mol, respectively. Invivo study results showed that the compound 5-Carbamoyl-2-formyl-1-[2-(4-nitrophenyl)-2-oxo-ethyl]-pyridinium gave 60% protection against epileptic seizures compared to 59% protection afforded by regular phenytoin. All of them met Lipinski's rule of five and had drug-likeness and drug score values of 0.55 and 0.8, respectively, making them chemically and functionally like phenytoin. According to the findings of the studies, the synthesized derivatives have the potential to be employed as a stepping stone in the development of novel anti-epileptic drugs.

Investigating the Anticancer Activity and Characterization of Bioactive Constituents of Moricandia sinaica (Boiss.) Boiss through In Vitro and In Silico Approaches in Triple-Negative Breast Cancer Cell Line

Resistance to chemotherapy and recurrence are major hurdles to treating hormone receptor-negative breast cancer. The crude extract and natural products obtained from medicinal plants are believed to be multitargeted and possess less toxicity as compared to synthetic compounds. The aerial parts and roots of Moricandia sinaica (Boiss.) Boiss were used to prepare the crude extracts in solvents of different polarities. Human breast cancer cell lines (MCF7 and MDA-MB-231), liver carcinoma (HepG2), and nontumorigenic cells of human origin (human umbilical vein endothelial cells (HUVEC)) were treated with a serial dilution of crude extracts obtained from the aerial and roots of Moricandia sinaica (Boiss.) Boiss. The methanol extract of the shoots exhibited a higher level of cytotoxicity against MDA-MB-231 cells than against any other cancer and nontumorigenic cells lines. Six new compounds were identified by gas chromatography–mass spectrophotometry analysis in the shoots extract of Moricandia sinaica (Boiss.) Boiss, and 2-Tridecen-1-ol was one of the major compounds that represent more than 35% of the extract. M-phase inducer phosphases 1 and 2 (CDC 25A and B) were identified as the specific protein target for 2-Tridecen-1-ol by the Swiss protein target prediction tool. In silico molecular docking showed the binding of 2-Tridecen-1-ol with CDC 25 B with a higher binding energy as compared to CDC 25A. The possible molecular mechanism of anticancer activity of Moricandia sinaica (Boiss.) Boiss in MDA-MB-231 breast cancer is through inhibition of M-phase inducer phosphatases 1 and 2 via 2-Tridecen-1-ol. Further investigations in breast cancer models are needed to explore the therapeutic potential of Moricandia sinaica (Boiss.) Boiss and 2-Tridecen-1-ol as an efficient remedy with a possibly less toxic approach to treat triple-negative breast cancer.

Characterization of squalene synthase gene from Gymnema sylvestre R. Br.

BackgroundSqualene synthase (SQS) is a rate-limiting enzyme necessary to produce pentacyclic triterpenes in plants. It is an important enzyme producing squalene molecules required to run steroidal and triterpenoid biosynthesis pathways working in competitive inhibition mode. Reports are available on information pertaining to SQS gene in several plants, but detailed information on SQS gene in Gymnema sylvestre R. Br. is not available. G. sylvestre is a priceless rare vine of central eco-region known for its medicinally important triterpenoids. Our work aims to characterize the GS-SQS gene in this high-value medicinal plant.ResultsCoding DNA sequences (CDS) with 1245 bp length representing GS-SQS gene predicted from transcriptome data in G. sylvestre was used for further characterization. The SWISS protein structure modeled for the GS-SQS amino acid sequence data had MolProbity Score of 1.44 and the Clash Score 3.86. The quality estimates and statistical score of Ramachandran plots analysis indicated that the homology model was reliable. For full-length amplification of the gene, primers designed from flanking regions of CDS encoding GS-SQS were used to get amplification against genomic DNA as template which resulted in approximately 6.2-kb sized single-band product. The sequencing of this product through NGS was carried out generating 2.32 Gb data and 3347 number of scaffolds with N50 value of 457 bp. These scaffolds were compared to identify similarity with other SQS genes as well as the GS-SQSs of the transcriptome. Scaffold_3347 representing the GS-SQS gene harbored two introns of 101 and 164 bp size. Both these intronic regions were validated by primers designed from adjoining outside regions of the introns on the scaffold representing GS-SQS gene. The amplification took place when the template was genomic DNA and failed when the template was cDNA confirmed the presence of two introns in GS-SQS gene in Gymnema sylvestre R. Br.ConclusionThis study shows GS-SQS gene was very closely related to Coffea arabica and Gardenia jasminoides and this gene harbored two introns of 101 and 164 bp size.

Novel pathogenic variant c.2714C>A (p. Thr905Lys) in the HK1 gene causing severe haemolytic anaemia with developmental delay in an Indian family

Hexokinase (EC 2.7.1.1, Adenosine Tri Phosphate (ATP): D-hexose-6-phosphotransferase) is a crucial regulatory enzyme of the glycolytic pathway (Embden-Meyerhof pathway). Hexokinase deficiency is associated with chronic non-spherocytic haemolytic anaemia (HA) with...

Alliin as a Natural Bioactive from Single Bulb Garlic (Allium sativum) for Nitric Oxide (NO) Increasing in Atherosclerotic Process Based on Insilico Screening

<p>Atherosclerosis is a chronic inflammatory disease in the arterial wall mediated by proinflammatory factor. Atherosclerosis cause heart disease and stroke is generally believed to be preventable. The single bulb garlic is one of the local plants known as the medicinal plant in Indonesia. Garlic contains <em>alliin</em> compounds as antiatherosclerotic agent. This study aimed to determine the potential of active compound of single bulb garlic as an antioxidant that improve endothelial function by increasing nitric oxide (NO) to increase vasodilation. The bioinformatics webserver used in this study are: Pubchem, Pharmmapper, Swiss Target Prediction, Superpred, Uniprot, and Protein Data Bank. Docking software using PyRx, PyMOL and Discovery Studio. Based on these steps, it was found that <em>alliin</em> interacts with nitric oxide synthase (NOS) through hydrogen and alkyl bonds. <em>Alliin</em> is effective as an inhibitor of NOS based on binding afinity (-5.4 kcal / mol) although no more negative than an aspirin inhibitor based on binding afinity (-6.5 kcal / mol). Based on the docking results, it is found that <em>alliin</em> is effective as a potential drug to decreased atherosclerosis.</p>

Estrogen alters the profile of the transcriptome in river snail Bellamya aeruginosa.

We evaluated the transcriptome dynamics of the freshwater river snail Bellamya aeruginosa exposed to 17β-estradiol (E2) using the Roche/454 GS-FLX platform. In total, 41,869 unigenes, with an average length of 586bp, representing 36,181 contigs and 5,688 singlets were obtained. Among them, 18.08, 36.85, and 25.47% matched sequences in the GenBank non-redundant nucleic acid database, non-redundant protein database, and Swiss protein database, respectively. Annotation of the unigenes with gene ontology, and then mapping them to biological pathways, revealed large groups of genes related to growth, development, reproduction, signal transduction, and defense mechanisms. Significant differences were found in gene expression in both liver and testicular tissues between control and E2-exposed organisms. These changes in gene expression will help in understanding the molecular mechanisms of the response to physiological stress in the river snail exposed to estrogen, and will facilitate research into biological processes and underlying physiological adaptations to xenoestrogen exposure in gastropods.

Transcriptomic response to estrogen exposure in the male Zhikong scallop, Chlamys farreri

The transcriptomes of Zhikong scallop exposed to 17β-estradiol were determined using the Roche/454. A total of 51,997 unigenes, representing 45,030 contigs and 6967 singlets were obtained. And 14,028, 19,798 and 14,981 of these unigenes were annotated from the non-redundant nucleic acid database, non-redundant protein database and Swiss protein database, respectively. A total of 10,699 unigenes were further annotated to biological processes (9080), molecular functions (8692) and cellular components (7829) using the GO, and 8945 unigenes were mapped to biological pathways including the metabolism (2862) and genetic information processing (2263). Most importantly, 16,692 unigenes and 18,686 unigenes in testis, and 10,492 unigenes and 13,186 unigenes in digestive gland were up-regulated significantly after exposure to 50 and 500ngE2/L; while 10,212 unigenes and 9409 unigenes in testis and 10,629 unigenes and 9463 unigenes in digestive gland were down-regulated. These valuable information provides insights into the mechanisms in invertebrate exposure to EDCs.

Abstract 3380: Comparison proteome analysis of scirrhous gastric carcinoma stem cell-like SP and hypoxia-resistant cell lines

Abstract Intro Cancer stem cells (CSCs) are considered to be responsible for cancer metastasis, and hypoxia is supposed to be an important regulator of CSCs differentiation. Furthermore, tumor hypoxia was reported to be associated with more aggressive tumor phenotypes such as high metastatic ability and resistance to various anti-cancer therapies which may lead to a poorer prognosis. This raises the question of whether there might be proteins representing similar alterations which are responsible for the correlation between hypoxic and CSCs phenotypes. We have established a diffuse-type of gastric carcinoma cell line (OCUM-12), and a hypoxia-resistant cancer cell line (OCUM-12/Hypo) cloned from parent OCUM-12 cells by continuous exposure to 1% oxygen. The side population (SP), as evaluated by a flow cytometric analysis using Hoechst 33342, has been known as CSC-rich population. To investigate and compare the proteomes of the hypoxic (OCUM-12/Hypo), stem cell (OCUM-12SP) and parent OCUM-12 diffuse-type gastric carcinoma cell lines, protein lysates from those cell lines were analyzed using QSTAR Elite LC MS/MS. Method Triplicate pooled samples (10 ug protein each) from stomach cancer cell lines were prepared and labeled with iTRAQ reagents. MS/MS data were searched against the Swiss Protein database (HUMAN) using ProteinPilot™ 2.0 software (AB Sciex). The Ingenuity Pathway analysis program was utilized, to assign biological significance, and to identify networks of interacting differentially expressed proteins, functional groups and pathways. Result In both OCUM-12SP and OCUM-12/Hypo cells, significant overexpression of Wnt signaling pathway was obvious. Furthermore, elevation of MTHFD1, a protein controlling histidine and purine nucleotide metabolism, cytokeratins 18 (CK18), and 19 (CK19) regulating cell cycle, apoptosis and angiogenesis, transcriptional regulator ANP32A, and cellular chaperones heat shock proteins A9 and STIP was observed as compared to the parent OCUM-12 cell line. Furthermore, anterior gradient homolog 2 (AGR2)was down-regulated in both cell lines. Conclusion Wnt signal, MTHFD1, CK18, CK19, ANP32A, HSPA9 and STIP, AGR2 might be responsible for the stem-like phenotype, hypoxia resistance and higher invasiveness of gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3380. doi:1538-7445.AM2012-3380

SwissPIT: An workflow‐based platform for analyzing tandem‐MS spectra using the Grid

The identification and characterization of peptides from MS/MS data represents a critical aspect of proteomics. It has been the subject of extensive research in bioinformatics resulting in the generation of a fair number of identification software tools. Most often, only one program with a specific and unvarying set of parameters is selected for identifying proteins. Hence, a significant proportion of the experimental spectra do not match the peptide sequences in the screened database due to inappropriate parameters or scoring schemes. The Swiss protein identification toolbox (swissPIT) project provides the scientific community with an expandable multitool platform for automated in-depth analysis of MS data also able to handle data from high-throughput experiments. With swissPIT many problems have been solved: The missing standards for input and output formats (A), creation of analysis workflows (B), unified result visualization (C), and simplicity of the user interface (D). Currently, swissPIT supports four different programs implementing two different search strategies to identify MS/MS spectra. Conceived to handle the calculation-intensive needs of each of the programs, swissPIT uses the distributed resources of a Swiss-wide computer Grid (http://www.swing-grid.ch).

Function assignment to Influenza B virus proteins using Support Vector Machine- A clue for vaccine development

Purpose: Identification of protein functions of Influenza B virus to understand the biological processes and networks of the virus which lead to novel way of diagnostics and vaccine designing. Methods: The protein sequences of Influenza B virus was retrieved from NCBI and Swiss Protein Databank. Prediction and analysis of different protein function family of proteins of Influenza B virus was done by the software, SVMProt, Support vector machine which classifies a protein into functional families from its primary sequence based on physico-chemical properties of amino acids.Results: The studies from SVMProt on Influenza B proteins suggest quite disparity of protein functions between structural and non-structural classes of proteins. ATP Binding cassette is a protein function shared by both the classes of proteins. Functions performed exclusively by Structural proteins are aptamer binding, these proteins belong to major facilitator family and transferasestransferring one carbon groups, whereas non-structural proteins performs functions like nuclear receptor and zinc-binding.Discussion and Conclusion: Protein function predictied by SVMProt is different for structural and non-structural protein of Influenza B virus, some of which may be responsible for virulence or pathogenicity of the virus and others for replication of the virus in the host. Functional classification of the proteins helps to facilitate the study of various biological processes and search for new therapeutic targets for vaccine designing.

Discovery and Validation of Novel Peptide Agonists for G-protein-coupled Receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

X-ray Structures of the Three Lactococcus lactis Dihydroxyacetone Kinase Subunits and of a Transient Intersubunit Complex

Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-angstroms crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-angstroms structure of the free DhaM dimer, and the 2.5-angstroms structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole Nepsilon2 of the DhaM His-10 and the beta-phosphate oxygen of ADP, between which the gamma-phosphate is transferred, is 4.9 angstroms. An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the gamma-phosphate in the transition state. The (betaalpha)4alpha fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.

Sensitization to wasp venom does not induce autoantibodies leading to infertility

Antigenic cross-reactivity has been described between the venom allergen (antigen 5) and mammalian testis proteins. Based on an allergen database we have previously shown that allergens can be represented by allergen motifs. A motif group was found containing venom antigen 5 sequences from different vespids. Using an optimized amino acid profile based on antigen 5 sequences for searching cross-reactive proteins, three human semen proteins belonging to the family of cysteine-rich secretory proteins (hCRISP) were found in the Swiss Protein database. To analyze antigenic cross-reactivity between antigen 5 and hCRISPs, antigen 5 from yellow jacket venom (Ves v 5) and two hCRISPs (CRISP-2 and -3) were chosen and produced as recombinant proteins in E. coli. A correlation was found between antibodies reacting with rVes v 5 and rhCRISP-2, -3 in a small human sera population indicating the presence of cross-reactive antibodies in human serum. Using intravenous immunoglobulin (IVIg), a therapeutic multidonor IgG preparation, cross-reactive antibodies were isolated that recognize rVes v 5, hCRISP-2 and -3 suggesting the presence of common epitopes between Ves v 5 and hCRISPs. However this cross-reactivity seems not to be linked to allergy to wasp venom as we could show no correlation between increasing CAP-class IgE level to wasp venom and IgG to sperm extract and hCRISPs. These data suggest that higher sensitization to wasp venom does not induce more antibodies against autoantigens and might not represent a higher risk to develop autoantibodies leading to infertility.

Retinal Pigment Epithelium Lipofuscin Proteomics

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

SwissPIT: a novel approach for pipelined analysis of mass spectrometry data

The identification and characterization of peptides from tandem mass spectrometry (MS/MS) data represents a critical aspect of proteomics. Today, tandem MS analysis is often performed by only using a single identification program achieving identification rates between 10-50% (Elias and Gygi, 2007). Beside the development of new analysis tools, recent publications describe also the pipelining of different search programs to increase the identification rate (Hartler et al., 2007; Keller et al., 2005). The Swiss Protein Identification Toolbox (swissPIT) follows this approach, but goes a step further by providing the user an expandable multi-tool platform capable of executing workflows to analyze tandem MS-based data. One of the major problems in proteomics is the absent of standardized workflows to analyze the produced data. This includes the pre-processing part as well as the final identification of peptides and proteins. The main idea of swissPIT is not only the usage of different identification tool in parallel, but also the meaningful concatenation of different identification strategies at the same time. The swissPIT is open source software but we also provide a user-friendly web platform, which demonstrates the capabilities of our software and which is available at http://swisspit.cscs.ch upon request for account.

Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators

BackgroundThe Fox gene family comprises a large and functionally diverse group of forkhead-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes.ResultsSystematic analysis of 458 protein sequences of the metazoan Fox family was performed to identify the presence of the engrailed homology-1 motif (eh1), a motif known to mediate physical interaction with transcriptional corepressors of the TLE/Groucho family. Greater than 50% of Fox proteins contain sequences with high similarity to the eh1 motif, including ten of the nineteen Fox subclasses (A, B, C, D, E, G, H, I, L, and Q) and Fox proteins of early divergent species such as marine sponge. The eh1 motif is not detected in Fox proteins of the F, J, K, M, N, O, P, R and S subclasses, or in yeast Fox proteins. The eh1-like motifs are positioned C-terminal to the winged helix DNA-binding domain in all subclasses except for FoxG proteins, which have an N-terminal motif. Two similar eh1-like motifs are found in the zebrafish FoxQ1 and in FoxG proteins of sea urchin and amphioxus. The identification of eh1-like motifs by manual sequence alignment was validated by statistical analyses of the Swiss protein database, confirming a high frequency of occurrence of eh1-like sequences in Fox family proteins. Structural predictions suggest that the majority of identified eh1-like motifs are short α-helices, and wheel modeling revealed an amphipathicity that supports this secondary structure prediction.ConclusionA search for eh1 Groucho interaction motifs in the Fox gene family has identified eh1-like sequences in greater than 50% of Fox proteins. The results predict a physical and functional interaction of TLE/Groucho corepressors with many members of the Fox family of transcriptional regulators. Given the functional importance of the eh1 motif in transcriptional regulation, our annotation of this motif in the Fox gene family will facilitate further study of the diverse transcriptional and regulatory roles of Fox family proteins.

Aberrant Folding of a Mutant Stat5b Causes Growth Hormone Insensitivity and Proteasomal Dysfunction

A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were approximately 3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.

Proteomics Reveal Cochlin Deposits Associated with Glaucomatous Trabecular Meshwork

The etiology of primary open angle glaucoma, a leading cause of age-related blindness, remains poorly defined, although elevated intraocular pressure (IOP) contributes to the disease progression. To better understand the mechanisms causing elevated IOP from aqueous humor circulation, we pursued proteomic analyses of trabecular meshwork (TM) from glaucoma and age-matched control donors. These analyses demonstrated that Cochlin, a protein associated with deafness disorder DFNA9, is present in glaucomatous but absent in normal TM. Cochlin was also detected in TM from the glaucomatous DBA/2J mouse preceding elevated IOP but found to be absent in three other mouse lines that do not develop elevated IOP. Histochemical analyses revealed co-deposits of Cochlin and mucopolysaccharide in human TM around Schlemm's canal, similar to that observed in the cochlea in DFNA9 deafness. Purified Cochlin was found to aggregate after sheer stress and to induce the aggregation of TM cells in vitro. Age-dependent in vivo increases in Cochlin were observed in glaucomatous TM, concomitant with a decrease in type II collagen, suggesting that Cochlin may disrupt the TM architecture and render components like collagen more susceptible to degradation and collapse. Overall, these observations suggest that Cochlin contributes to elevated IOP in primary open angle glaucoma through altered interactions within the TM extracellular matrix, resulting in cell aggregation, mucopolysaccharide deposition, and significant obstruction of the aqueous humor circulation.

Trypanosome Cyclic Nucleotide Phosphodiesterase 2B Binds cAMP through Its GAF-A Domain

Trypanosoma brucei, the causative agent of sleeping sickness in humans and livestock, expresses at least three cAMP-specific class I phosphodiesterases (PDEs), all of which are essential for survival of the parasite. These PDEs have either one or two N-terminal GAF domains, which in other proteins function as signaling domains. However, neither the functional roles nor ligands for these domains in trypanosome PDEs are known. The present study shows that TbPDE2B, which contains two tandem GAF domains, binds cAMP with high affinity through its GAF-A domain. A purified recombinant N terminus + GAF-A domain binds cAMP with an affinity (Ki) of approximately 16 nM. It also binds cGMP but with a 15-fold lower affinity of approximately 275 nM. The TbPDE2B holoenzyme has a somewhat lower affinity (approximately 55 nM) for cAMP but a greatly lower affinity (approximately 10 microM) for cGMP. This suggests that both the selectivity and affinity for a ligand can be determined not only by the nature of the binding domain but also by the adjacent domains. Additionally, binding of cAMP to the holoenzyme showed positive cooperativity, with a Hill coefficient value of 1.75. However, binding of cGMP to the holoenzyme did not show any cooperativity, suggesting differences in the conformational changes caused by binding of these two cyclic nucleotides with the protein. Point mutation of a key predicted binding site residue (T317A) resulted in a complete loss of high affinity cAMP binding. This mutation increased the apparent Km of the mutant enzyme for substrate without altering the Vmax. A truncated catalytic domain construct of TbPDE2B also exhibited an increased Km, strongly suggesting that cAMP binding to the GAF-A domain can regulate TbPDE2B by allowing the full activity of the enzyme to be expressed. These properties of the GAF-A domain of TbPDE2B thus suggest that it could be a new target for anti-trypanosomal drugs.