Small ubiquitin-like modifier (SUMO) modification of E1 Cys domain inhibits E1 Cys domain enzymatic activity.

Although it is well established that ubiquitin-like modifications are tightly regulated, it has been unclear how their E1 activities are controlled. In this study, we found that the SAE2 subunit of the small ubiquitin-like modifier (SUMO) E1 is autoSUMOylated at residue Lys-236, and SUMOylation was catalyzed by Ubc9 at several additional Lys residues surrounding the catalytic Cys-173 of SAE2. AutoSUMOylation of SAE2 did not affect SUMO adenylation or formation of E1·SUMO thioester, but did significantly inhibit the transfer of SUMO from E1 to E2 and overall SUMO conjugations to target proteins due to the altered interaction between E1 and E2. Upon heat shock, SUMOylation of SAE2 was reduced, which corresponded with an increase in global SUMOylation, suggesting that SUMOylation of the Cys domain of SAE2 is a mechanism for "storing" a pool of E1 that can be quickly activated in response to environmental changes. This study is the first to show how E1 activity is controlled by post-translational modifications, and similar regulation likely exists across the homologous E1s of ubiquitin-like modifications.

Sumoylation of SAE2 C terminus regulates SAE nuclear localization.

SUMOylation occurs predominantly in the nucleus, but non-nuclear proteins can also be SUMOylated. It is unclear how intracellular trafficking of the SUMOylation enzymes is regulated to catalyze SUMOylation in different cellular compartments. Here we report that the SAE2 subunit of human SUMO activation enzyme (SAE) underwent rapid nucleocytoplasmic shuttling and its nuclear accumulation depended on SUMO modification at the C terminus. The SUMOylation sites included three Lys residues on the bipartite nuclear localization sequence (NLS) and two Lys residues outside of but adjacent to the NLS, and their SUMOylation was catalyzed by Ubc9. Because SAE2 forms a tight heterodimer with SAE1 and it controls the trafficking of the heterodimer, this study has identified the mechanism used to localize SAE to the nucleus. Similar mechanisms are likely to exist for other proteins that depend on SUMOylation for nuclear localization.

Proteomic profiling following immunoaffinity capture of high-density lipoprotein: association of acute-phase proteins and complement factors with proinflammatory high-density lipoprotein in rheumatoid arthritis.

OBJECTIVE: To identify protein biomarkers associated with proinflammatory high-density lipoprotein (HDL) in patients with active rheumatoid arthritis (RA) by proteomic analysis. METHODS: Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze proteins associated with immunoaffinity-purified HDL from plasma obtained from 2 sets of RA patients, 1 with antiinflammatory HDL and 1 with proinflammatory HDL. Proteins were fractionated by Offgel electrophoresis and analyzed using an LC-MS/MS system equipped with a high-capacity high-performance liquid chromatography chip incorporating C18 reverse-phase trapping and analytical columns. Sandwich enzyme-linked immunosorbent assays were used to validate the association between select proteins and proinflammatory HDL in a second cohort of RA patients. RESULTS: Seventy-eight proteins were identified in the HDL complexes. The levels of 12 proteins were significantly increased in RA patients with proinflammatory HDL compared to RA patients with antiinflammatory HDL. These proteins included the acute-phase proteins apolipoprotein J, fibrinogen, haptoglobin, serum amyloid A, and complement factors (B, C3, and C9). The associations between proinflammatory HDL and 4 of the proteins were validated in a second RA cohort. CONCLUSION: Our findings indicate that proinflammatory HDL in patients with RA contains a significantly altered proteome, including increased amounts of acute-phase proteins and proteins involved in the complement cascade. These findings suggest that HDL is significantly altered in the setting of chronic inflammation in active RA, with resultant loss of its antiinflammatory function. The characterization of the biomarkers described herein may identify novel molecular connections that contribute to the higher risk of cardiovascular disease in RA patients.

Proteomic analysis of surface and endosomal membrane proteins from the avian LMH epithelial cell line.

Proteins at the cell surface and within the endocytic pathway are increasingly being recognized for their roles in a wide variety of intercellular interactions. Here we used the inherent hydrophobicity and N-glycosylation of membrane proteins to enrich these proteins from the surface and endosome of avian LMH epithelial cells for mass spectrometric analysis. The cycling of many different types of proteins from the cell surface into the endosome and sometimes back to the surface again makes it appropriate to analyze these two membranous cellular components together. Stringent searches of the International Protein Index (IPI) entries for Gallus gallus identified 318 unique integral membrane proteins (IMPs) (201 bearing N-glycosylation sites), 265 unique membrane-associated proteins (MAPs), and an additional group of 784 non-membrane proteins (NMPs) among TX-114 detergent and aqueous phase-enriched proteins. Capture of N-glycosylated tryptic peptides revealed 36 additional glycoproteins most of which were CD antigens, receptors, and molecules for cell adhesion and immune response. IMPs and MAPs present at the surface and within the endosome included proteins involved in transport (255), metabolism (285), communication (108), adhesion (47), and immune responses (42). Among these were 355 putative uncharacterized and hypothetical IMPs, MAPs, and NMPs for which highly similar annotated sequences were found in standard protein-protein BLAST searches.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors.

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme.

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Deposition of exon-skipping splice isoform of human retinal G protein-coupled receptor from retinal pigment epithelium into Bruch's membrane.

PURPOSE: Human retina and retinal pigment epithelium (RPE) express a relatively abundant mRNA that encodes an extraneous splice isoform of the RPE retinal G protein-coupled receptor (RGR) opsin. In this study, we investigate this exon-skipping RGR splice isoform (RGR-d) in separated neural retina and RPE cells of human donors of various ages. METHODS: We used mass spectrometry, sensitive western blot assay, immunohistochemical localization and real-time RT-PCR to analyze RGR-d. RESULTS: Western blot assay detected the RGR-d protein in the neural retina of all donors analyzed. Mass spectrometric analysis of the immunoreactive proteins independently confirmed the presence of RGR-d. In contrast, RGR-d protein in the RPE of most donors was barely detectable by western blot assay, even though expression of RGR-d mRNA was confirmed by amplification of RGR-d transcripts in both the RPE and neural retina. Quantitative real-time RT-PCR assays showed that RGR-d/RGR mRNA transcript ratios were about 0.17 and about 0.33 in the RPE and neural retina, respectively. Immunohistochemical localization studies revealed that the RGR-d epitope was present near the basal boundary of RPE cells and primarily in the extracellular areas of Bruch's membrane, adjacent choriocapillaris, and intercapillary region of both young and older donors. Positive immunostaining was seen in the drusen of older individuals. CONCLUSIONS: The RGR-d protein is a common mutant form of human RGR that can be identified in donor eyes by mass spectrometry. These results indicate that after RGR-d is synthesized, the RGR-d epitope is released at the basal surface of the RPE and deposited into Bruch's membrane in human eyes throughout adult life.

Median filter algorithm for estimating the threshold of detection on custom protein arrays.

We constructed protein arrays according to a titration design to estimate the assay sensitivities over varying concentrations of flu vaccine and human immunoglobulin G (IgG). After imaging, we considered the problem of appropriately distinguishing background noise from foreground signal. We applied the median filter smoothing technique and estimated the differences of the observed signal compared to the smoothed signal. If the absolute value of the difference was large, the feature was easily detectable, indicating that the spot did not blend with its surrounding neighbors. After estimating the residuals, we applied thresholding algorithms to estimate the limits of detection for each assay. At sufficiently large smoothing spans, our median filter approach performed as well or better than visual inspection and two other competing analysis methods. This suggests that a median filter approach has utility in high-throughput arrays where visual inspection is impractical.

DNA affinity capture and protein profiling by SELDI-TOF mass spectrometry: effect of DNA methylation.

We report here that surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry, as performed on a Ciphergen Biosystems ProteinChip System, can be used in conjunction with DNA affinity capture (DACA) to study specific DNA-protein binding. Using DNA molecules bound to a surface, sequence-specific interactions can be detected as demonstrated by a mutation affecting the binding profile for TBP, a transcription factor. Also, a comparison between methylated and unmethylated promoter-containing DNA fragments shows numerous binding profile differences over a mass range extending to >60 kDa. The binding of several proteins is inhibited by methylation of the DNA.

Solution structure of the C-terminal domain of Ku80 suggests important sites for protein-protein interactions.

The solution structure of Ku80 CTD from residue 566 to 732 has been solved in order to gain insights into the mechanisms of its interactions with other proteins. The structure reveals a topology similar to several common scaffolds for protein-protein interactions, in the absence of significant sequence similarity to these proteins. Conserved surface amino acid residues are clustered on two main surface areas, which are likely involved in mediating interactions between Ku80 and other proteins. The Ku70/Ku80 heterodimer has been shown to be involved in at least three processes, nonhomologous end joining, transcription, and telomere maintenance, and thus it needs to interact with different proteins involved in these different processes. The three-dimensional structure of the Ku80 C-terminal domain and the availability of NMR chemical shift assignments provide a basis for further investigation of the interactions between Ku80 and other proteins in these Ku-dependent cellular functions.

Photoreceptor mitochondrial tyrosine nitration in experimental uveitis.

PURPOSE: In experimental autoimmune uveitis (EAU), phagocytes are thought to be the primary cells in the initiation and maintenance of pathologic tissue damage through the release of cytotoxic agents. Recently, the presence of nitric oxide synthase has been shown in mammalian mitochondria. In this study, the effect of mitochondrial peroxynitrite on the modification of cellular proteins was evaluated in the early phase of uveitis, before the infiltration of leukocytes. METHODS: Tyrosine nitration in proteins was detected by UV/Vis (visible) absorption and Western blot analysis. The identity of the nitrated protein was obtained by liquid chromatography-tandem mass spectrometry. The release of cytochrome c was assessed in whole retinal extract and in isolated mitochondria. The protein nitration in the inflamed retina was also localized by immunohistochemistry. RESULTS: Before the leukocyte infiltration in the early phase of EAU, the mitochondria-originated peroxynitrite initiated the inflammatory insult by specifically nitrating three mitochondrial proteins. In vitro nitration of the control retina by peroxynitrite donor resulted in nonspecific nitration of all major retinal proteins. After nitration, cytochrome c was displaced from its original binding site in the respiratory chain. Further, the nitration appeared to commence in the early phase of inflammation, on postimmunization day 5, long before the peak of inflammation on day 14. Immunohistochemically, tyrosine-nitrated proteins were localized exclusively in the photoreceptor inner segments, which are known to be densely populated with mitochondria. CONCLUSIONS: These data indicate that mitochondrial proteins are the prime targets of inactivation by the mitochondrial peroxynitrite and that photoreceptor mitochondria initiate the subsequent irreversible retinal damage in experimental uveitis.

Qscore: an algorithm for evaluating SEQUEST database search results.

A scoring procedure is described for measuring the quality of the results for protein identifications obtained from spectral matching of MS/MS data using the Sequest database search program. The scoring system is essentially probabilistic and operates by estimating the probability that a protein identification has come about by chance. The probability is based on the number of identified peptides from the protein, the total number of identified peptides, and the fraction of distinct tryptic peptides from the database that are present in the identified protein. The score is not strictly a probability, as it also incorporates information about the quality of the individual peptide matches. The result of using Qscore on a large test set of data was similar to that achieved using approaches that validate individual spectral matches, with only a narrow overlap in scores between identified proteins and false positive matches. In direct comparison with a published method of evaluating Sequest results, Qscore was able to identify an equivalent number of proteins without any identifiable false positive assignments. Qscore greatly reduces the number of Sequest protein identifications that have to be validated manually.

Synthesis of biologically active canine CCK-58.

The carboxyl terminal octapeptide of cholecystokinin (CCK-8) has been hypothesized to account for the bioactivity of all the molecular forms of cholecystokinin. However, the physiological relevance of CCK-58 has not been rigorously examined because of the lack of sufficient amounts of the peptide and concerns about inactivation of natural peptides during their purification. Therefore, canine-sulfated CCK-58 was synthesized and conditions determined for its unblocking and purification that preserved the sulfated tyrosine. Synthetic CCK-58 was indistinguishable from natural CCK-58 by amino acid analysis and by mass spectrometry. Synthetic CCK-58 and CCK-8 have different patterns of pancreatic stimulation: both caused a dose-related increase in amylase release, while only CCK-58 stimulated bile-pancreatic output volume. Thus, CCK-58 and CCK-8 are biased agonists at the CCK-A receptor (they have distinct patterns of action mediated by the same receptor). Previous work has demonstrated that the identical carboxyl termini of CCK-8 and CCK-58 have different solution conformations. Taken together, the physiological and structural results support the hypothesis that different carboxyl terminal conformations of CCK-58 and CCK-8 alter the expression of their biological activity.

Rat progastrin processing yields peptides with altered potency at the CCK-B receptor.

Details of prohormone processing patterns are revealed by purification and characterization of molecular forms stored in the tissues where the hormones are expressed. Molecular forms of rat gastrin were purified from antral extracts by gel permeation, anion exchange, and reverse-phase HPLC. Amidated and glycine-extended gastrins were detected with specific antisera and their structures determined by mass spectrometry. In rats, the only form shorter than gastrin-17 observed contained 16 amino acids. These data suggest that two enzymes process the amino terminus of gastrin-17. Pyrrolidone carboxylic acid peptidase removes the amino terminal pyrrolidone carboxylic acid (pyroGlu), forming gastrin-16. In mammals other than rat, gastrin-16 is then cleaved by dipeptidyl peptidase IV to form gastrin-14. In rat, this reaction does not take place because of proline residues Pro(2)-Pro(3)- in gastrin-16. Gastrin-16 is found in sulfated and nonsulfated forms and comprises 28% of the total gastrin immunoreactivity. Glycine-extended forms of gastrin-16 and gastrin-17 comprises 45% of the total gastrin immunoreactivity. The sulfated forms of gastrin-16 and gastrin-17 bind to the CCK-B receptor transfected into CHO cells with 10-fold higher affinity than the nonsulfated forms of these peptides. Therefore, processing of rat progastrin may modulate the expression of gastrin biological activity.

Sequence analysis and feeding responses evoked by the large molecular form of gastrin releasing peptide (GRP) in the rat GRP-29.

Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03 nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41 nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.

Introducing samples directly into electrospray ionization mass spectrometers by direct infusion using a nanoelectrospray interface.

Procedures are described for constructing and using a microscale electrospray interface for direct infusion of samples into mass spectrometers. The sensitivity of the nanospray interface is a result of greatly reducing the flow of sample solution while preserving the analyte signal intensity. The described methodology provides a simple and robust way to analyze individual purified peptide and protein samples, i.e., samples that do not require liquid chromatography separation.

MHC class I target recognition, immunophenotypes and proteomic profiles of natural killer cells within the spleens of day-14 chick embryos.

Chicken natural killer (NK) cells are not well defined, so little is known about the molecular interactions controlling their activity. At day 14 of embryonic development, chick spleens are a rich source of T-cell-free CD8αα(+), CD3(-) cells with natural killing activity. Cell-mediated cytotoxicity assays revealed complex NK cell discrimination of MHC class I, suggesting the presence of multiple NK cell receptors. Immunophenotyping of freshly isolated and recombinant chicken interleukin-2-stimulated d14E CD8αα(+) CD3(-) splenocytes provided further evidence for population heterogeneity. Complex patterns of expression were found for CD8α, chB6 (Bu-1), CD1-1, CD56 (NCAM), KUL01, CD5, and CD44. Mass spectrometry-based proteomics revealed an array of NK cell proteins, including the NKR2B4 receptor. DAVID and KEGG analyses and additional immunophenotyping revealed NK cell activation pathways and evidence for monocytes within the splenocyte cultures. This study provides an underpinning for further investigation into the specificity and function of NK cells in birds.

Alkylation of cysteine 468 in Stat3 defines a novel site for therapeutic development.

Stat3 is a latent transcription factor that promotes cell survival and proliferation and is often constitutively active in multiple cancers. Inhibition of Stat3 signaling pathways suppresses cell survival signals and leads to apoptosis in cancer cells, suggesting direct inhibition of Stat3 function is a viable therapeutic approach. Herein, we identify a small molecule, C48, as a selective Stat3-family member inhibitor. To determine its mechanism of action, we used site-directed mutagenesis and multiple biochemical techniques to show that C48 alkylates Cys468 in Stat3, a residue at the DNA-binding interface. We further demonstrate that C48 blocks accumulation of activated Stat3 in the nucleus in tumor cell lines that overexpress active Stat3, leading to impressive inhibition of tumor growth in mouse models. Collectively, these findings suggest Cys468 in Stat3 represents a novel site for therapeutic intervention and demonstrates the promise of alkylation as a potentially effective chemical approach for Stat3-dependent cancers.

Comparative analyses of differentially induced T-cell receptor-mediated phosphorylation pathways in T lymphoma cells.

Activation of T lymphoma cells expressing Syk, but not ZAP-70 tyrosine kinase, has been shown to negatively regulate cell activation and activation-induced cell death (AICD), perhaps due to differential induction of tyrosine phosphorylation modified proteins. To better understand the role of these proteins and their associated molecules/pathways, we studied a previously described model of T lymphoma cells expressing either a kinase-activated chimeric Syk or ZAP-70 genetically linked to T-cell receptor (TCR) ζ chain (Z/Syk or Z/ZAP cells, respectively). To help identify molecules and pathways linked to cell activation or AICD, a comparative semi-quantitative proteomics-based approach was utilized to analyze tyrosine-phosphorylated protein immunoprecipitates from two-minute short-term activated Z/Syk or Z/ZAP cells. Using the resulting bioinformatics data-sets, we identified several differentially immunoprecipitated proteins that could be validated biochemically. More tyrosine-phosphorylated and phosphotyrosine-associated proteins were found in Z/Syk than in Z/ZAP cells. Proteins involved in different unique functional pathways were induced in these cells and showed altered intermolecular interactions in varied pathways. Remarkably, 41% of differentially identified proteins in Z/Syk cells belonged to cell cycle or vesicle/trafficking pathways. In contrast, 21% of such proteins in Z/ZAP cells belonged to metabolism pathways. Therefore, molecular pathways involved in post-translational modifications linked to distinct cellular/physiological functions are differentially activated, which may contribute to varied activation and AICD responses of these cells. In summary, we identified proteins belonging to novel differentially activated pathways involved in TCR-mediated signaling, which may be targets for regulating activation and AICD of T lymphoma cells and for potential cancer therapy.

Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-B haplotypes associated with opposite responses to Marek's disease.

In the chicken, resistance to lymphomas that form following infection with oncogenic strains of Marek's herpesvirus is strongly linked to the major histocompatibility complex (MHC)-B complex. MHC-B21 haplotype is associated with lower tumor-related mortality compared to other haplotypes including MHC-B13. The single, dominantly expressed class I gene (BF2) is postulated as responsible for the MHC-B haplotype association. We used mass spectrometry to identify peptides and structural modeling to define the peptide binding preferences of BF2 2101 and BF2 1301 proteins. Endogenous peptides (8-12 residues long) were eluted from affinity-purified BF2 2101 and BF2 1301 proteins obtained from transduced cDNA expressed in RP9 cells, hence expressed in the presence of heterologous TAP. Sequences of individual peptides were identified by mass spectrometry. BF2 2101 peptides appear to be tethered at the binding groove margins with longer peptides arching out but selected by preferred residues at positions P3, P5, and P8: X-X-[AVILFP]-X((1-5))-[AVLFWP]-X((2-3))-[VILFM]. BF2 1301 peptides appear selected for residues at P2, P3, P5, and P8: X-[DE]-[AVILFW]-X((1-2))-[DE]-X-X-[ED]-X((0-4)). Some longer BF2 1301 peptides likely also arch out, but others are apparently accommodated by repositioning of Arg83 so that peptides extend beyond the last preferred residue at P8. Comparisons of these peptides with earlier peptides derived in the presence of homologous TAP transport revealed the same side chain preferences. Scanning of Marek's and other viral proteins with the BF2 2101 motif identified many matches, as did the control human leukocyte antigen A 0201 motif. The BF2 1301 motif is more restricting suggesting that this allele may confer a selective advantage only in infections with a subset of viral pathogens.