Brian D. Halligan

Comparative proteomic analysis of PAI-1 and TNF-alpha-derived endothelial microparticles

Endothelium‐derived microparticles (EMPs) are small vesicles released from endothelial cells in response to cell injury, apoptosis, or activation. Elevated concentrations of EMPs have been associated with many inflammatory and vascular diseases. EMPs also mediate long range signaling and alter downstream cell function. Unfortunately, the molecular and cellular basis of microparticle production and downstream cell function is poorly understood. We hypothesize that EMPs generated by different agonists will produce distinct populations of EMPs with unique protein compositions. To test this hypothesis, different EMP populations were generated from human umbilical vein endothelial cells by stimulation with plasminogen activator inhibitor type 1 (PAI‐1) or tumor necrosis factor‐alpha (TNF‐α) and subjected to proteomic analysis by LC/MS. We identified 432 common proteins in all EMP populations studied. Also identified were 231 proteins unique to control EMPs, 104 proteins unique to PAI‐1 EMPs and 70 proteins unique to TNF‐α EMPs. Interestingly, variations in protein abundance were found among many of the common EMP proteins, suggesting that differences exist between EMPs on a relative scale. Finally, gene ontology (GO) and KEGG pathway analysis revealed many functional similarities and few differences between the EMP populations studied. In summary, our results clearly indicate that EMPs generated by PAI‐1 and TNF‐α produce EMPs with overlapping but distinct protein compositions. These observations provide fundamental insight into the mechanisms regulating the production of these particles and their physiological role in numerous diseases.

ProMoST (Protein Modification Screening Tool): a web-based tool for mapping protein modifications on two-dimensional gels

ProMoST is a flexible web tool that calculates the effect of single or multiple posttranslational modifications (PTMs) on protein isoelectric point (pI) and molecular weight and displays the calculated patterns as two-dimensional (2D) gel images. PTMs of proteins control many biological regulatory and signaling mechanisms and 2D gel electrophoresis is able to resolve many PTM-induced isoforms, such as those due to phosphorylation, acetylation, deamination, alkylation, cysteine oxidation or tyrosine nitration. These modifications cause changes in the pI of the protein by adding, removing or changing titratable groups. Proteins differ widely in buffering capacity and pI and therefore the same PTMs may give rise to quite different patterns of pI shifts in different proteins. It is impossible by visual inspection of a pattern of spots on a gel to determine which modifications are most likely to be present. The patterns of PTM shifts for different proteins can be calculated and are often quite distinctive. The theoretical gel images produced by ProMoST can be compared to the experimental 2D gel results to implicate probable PTMs and focus efforts on more detailed study of modified proteins. ProMoST has been implemented as cgi script in Perl available on a WWW server at http://proteomics.mcw.edu/promost.

Low Cost, Scalable Proteomics Data Analysis Using Amazon's Cloud Computing Services and Open Source Search Algorithms

One of the major difficulties for many laboratories setting up proteomics programs has been obtaining and maintaining the computational infrastructure required for the analysis of the large flow of proteomics data. We describe a system that combines distributed cloud computing and open source software to allow laboratories to set up scalable virtual proteomics analysis clusters without the investment in computational hardware or software licensing fees. Additionally, the pricing structure of distributed computing providers, such as Amazon Web Services, allows laboratories or even individuals to have large-scale computational resources at their disposal at a very low cost per run. We provide detailed step-by-step instructions on how to implement the virtual proteomics analysis clusters as well as a list of current available preconfigured Amazon machine images containing the OMSSA and X!Tandem search algorithms and sequence databases on the Medical College of Wisconsin Proteomics Center Web site ( http://proteomics.mcw.edu/vipdac ).

Antiviral Inhibition Targeting the HCMV Kinase pUL97 Requires pUL27-Dependent Degradation of Tip60 Acetyltransferase and Cell-Cycle Arrest

Infection with the β-herpesvirus human cytomegalovirus (HCMV) is lifelong, causing limited disease in healthy adults, but life threatening in immunocompromised individuals. The viral kinase pUL97, a functional ortholog of cellular cyclin-dependent kinases (CDKs), is critical for HCMV replication and a target for antiviral drug development. Upon kinase inhibition, drug-resistant strains emerge with mutations in UL27, an HCMV gene of unknown function. Using a proteomics approach, we discovered that pUL27 is necessary and sufficient to degrade Tip60, a host acetyltransferase and interacting partner of HIV Tat. Consistent with this, the expression of Tat restored antiviral inhibition of an otherwise resistant HCMV strain. The functional consequence of Tip60 degradation was the induction of the CDK inhibitor p21(Waf1/Cip1) and cell-cycle arrest, representing changes necessary for the antiviral effects of pUL97 inhibition. Consequently, either increasing p21(Waf1/Cip1) expression or decreasing Tip60 levels improved the antiviral activity of the HCMV kinase inhibitor maribavir.

Mitochondrial proteomic analysis reveals deficiencies in oxygen utilization in medullary thick ascending limb of Henle in the Dahl salt-sensitive rat

The renal medullary thick ascending limb (mTAL) of the Dahl salt-sensitive (SS) rat is the site of enhanced NaCl reabsorption and excess superoxide production. In the present studies we isolated mitochondria from mTAL of SS and salt-resistant control strain SS.13(BN) rats on 0.4 and 8% salt diet for 7 days and performed a proteomic analysis. Purity of mTAL and mitochondria isolations exceeded 93.6 and 55%, respectively. Using LC/MS spectral analysis techniques we identified 96 mitochondrial proteins in four biological mTAL mitochondria samples, run in duplicate, as defined by proteins with a false discovery rate <5% and scan count ≥2. Seven of these 96 proteins, including IDH2, ACADM, SCOT, Hsp60, ATPA, EFTu, and VDAC2 were differentially expressed between the two rat strains. Oxygen consumption and high-resolution respirometry analyses showed that mTAL cells and the mitochondria in the outer medulla of SS rats fed high-salt diet exhibited lower rates of oxygen utilization compared with those from SS.13(BN) rats. These studies advance the conventional proteomic paradigm of focusing exclusively upon whole tissue homogenates to a focus upon a single cell type and specific subcellular organelle. The results reveal the importance of a largely unexplored role for deficiencies of mTAL mitochondrial metabolism and oxygen utilization in salt-induced hypertension and renal medullary oxidative stress.

Candidate Urinary Biomarker Discovery in Ureteropelvic Junction Obstruction: A Proteomic Approach

PURPOSE Ureteropelvic junction obstruction may either worsen and require surgery, improve or remain stable. It may take upward of 3 years for the natural history to unfold. Urinary proteome analysis using capillary electrophoresis mass spectrometry has been shown to differentiate between normal infants and those with ureteropelvic junction obstruction. We sought to confirm these findings using liquid chromatography/nano-spray mass spectrometry to examine the urinary proteome in patients with unilateral grade IV ureteropelvic junction obstruction compared to age matched healthy infants. MATERIALS AND METHODS Urine specimens were obtained from 21 healthy infants with normal maternal/fetal ultrasound and 25 infants with grade IV unilateral ureteropelvic junction obstruction. Specimens were prepared using standard methods and subjected to liquid chromatography/tandem mass spectrometry analysis. Normalized data were annotated using the IPA(R) knowledge platform. RESULTS There were 31 proteins significantly different in their level of abundance at 1 to 6 months, and 18 at 7 to 12 months compared to age matched controls. These proteins clustered into major functional networks. All of the biomarkers previously reported in clinical studies of ureteropelvic junction obstruction were observed with the notable exception of transforming growth factor-beta1. CONCLUSIONS These results confirm the presence of significant differences in the urinary proteome in unilateral ureteropelvic junction obstruction compared to age matched normal individuals. This study adds new information about levels of abundance of specific proteins and peptides in ureteropelvic junction obstruction, which may allow for better classification of disease subgroups and help to establish improved indications for the early selection of surgical candidates based on urinary protein biomarkers.

Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase

The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had been previously purified from the conditioned culture media of porcine articular cartilage. Protein sequencing of an internal 61-kDa proteolytic fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids. This sequence was used to amplify a DNA fragment, which was used as a probe to clone the gene encoding the 61-kDa NTPPHase from a porcine chondrocyte cDNA library. DNA sequence analysis showed the cDNA insert to be 2509 bp, corresponding to a predicted open reading frame (ORF) encoding 599 amino acids. The 26 N-terminal amino acids of the 61-kDa NTPPHase were located within the ORF immediately downstream of a putative protease recognition region, RRKRR. This is consistent with this cDNA insert representing an internal proteolytic fragment of the full length 127-kDa NTPPHase. BLAST and FASTA analysis confirmed that the deduced amino acid sequence of 61-kDa NTPPHase was unique and did not possess a high degree of homology to sequence in the non-redundant protein and nucleotide databases. Proteins that possess limited homology (< 17%) with the 61-kDa NTTPPHase include several prokaryotic and eukaryotic ATP pyrophosphate-lyases (adenylate cyclase). Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61-kDa NTPPHase hybridized to a single 4.0-kb RNA transcript. This DNA probe also hybridized to a single species of human chondrocyte RNA. Expression of a 61-kDa protein was detected by coupled in-vitro transcription/translation. Western blot analysis of this in-vitro transcription/translation reaction detected a 61-kDa protein, using an antibody raised against the peptide sequence that was originally used to clone the 61-kDa NTPPHase. These data indicate the successful in-vitro cloning and expression of the porcine chondrocyte 61-kDa NTPPHase. Future studies that utilize the gene encoding the 61-kDa NTPPHase may allow the characterization of the role of NTPPHase in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.

Visualize: a free and open source multifunction tool for proteomics data analysis.

A major challenge in the field of high‐throughput proteomics is the conversion of the large volume of experimental data that is generated into biological knowledge. Typically, proteomics experiments involve the combination and comparison of multiple data sets and the analysis and annotation of these combined results. Although there are some commercial applications that provide some of these functions, there is a need for a free, open source, multifunction tool for advanced proteomics data analysis. We have developed the Visualize program that provides users with the abilities to visualize, analyze, and annotate proteomics data; combine data from multiple runs, and quantitate differences between individual runs and combined data sets. Visualize is licensed under GNU GPL and can be downloaded from http://proteomics.mcw.edu/visualize. It is available as compiled client‐based executable files for both Windows and Mac OS X platforms as well as PERL source code.

ProMoST: a tool for calculating the pI and molecular mass of phosphorylated and modified proteins on two-dimensional gels.

Protein modifications such as phosphorylation are often studied by two-dimensional gel electrophoresis, since the perturbation in the proteins pI value is readily detected by this method. It is important to be able to calculate the changes in the pI values that specific post-translational modifications cause and to visualize how these changes will effect protein migration on 2D gels. To address this need, we have developed ProMoST. ProMoST is a freely accessible Web-based application that calculates and displays the mass and pI values for either proteins in the NCBI database identified by accession number or from submitted FASTA format sequence.

Human Cytomegalovirus pUL29/28 and pUL38 Repression of p53-Regulated p21CIP1 and Caspase 1 Promoters during Infection

ABSTRACT During infection by human cytomegalovirus (HCMV), the tumor suppressor protein p53, which promotes efficient viral gene expression, is stabilized. However, the expression of numerous p53-responsive cellular genes is not upregulated. The molecular mechanism used to manipulate the transcriptional activity of p53 during infection remains unclear. The HCMV proteins IE1, IE2, pUL44, and pUL84 likely contribute to the regulation of p53. In this study, we used a discovery-based approach to identify the protein targets of the HCMV protein pUL29/28 during infection. Previous studies have demonstrated that pUL29/28 regulates viral gene expression by interacting with the chromatin remodeling complex NuRD. Here, we observed that pUL29/28 also associates with p53, an additional deacetylase complex, and several HCMV proteins, including pUL38. We confirmed the interaction between p53 and pUL29/28 in both the presence and absence of infection. HCMV pUL29/28 with pUL38 altered the activity of the 53-regulatable p21CIP1 promoter. During infection, pUL29/28 and pUL38 contributed to the inhibition of p21CIP1 as well as caspase 1 expression. The expression of several other p53-regulating genes was not altered. Infection using a UL29-deficient virus resulted in increased p53 binding and histone H3 acetylation at the responsive promoters. Furthermore, expression of pUL29/28 and its interacting partner pUL38 contributed to an increase in the steady-state protein levels of p53. This study identified two additional HCMV proteins, pUL29/28 and pUL38, which participate in the complex regulation of p53 transcriptional activity during infection.