Jeffrey A. Kowalak

The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 Study Relative Protein Quantitation

The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of Biomolecular Resource Facilities Proteomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 Proteomics Research Group study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC-, and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies used.

Regulation of α-synuclein expression by poly (ADP ribose) polymerase-1 (PARP-1) binding to the NACP-Rep1 polymorphic site upstream of the SNCA gene

Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1s catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1s catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.

Nitration and Inactivation of IDO by Peroxynitrite

IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

Studies towards neoglycoconjugates from the monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa using the diethyl squarate reagent

The effect of reaction time, concentration and molar excess of hapten upon the efficiency of the conjugation of carbohydrates to proteins using the diethyl squarate reagent has been studied using chicken serum albumin (CSA) as the carrier protein and a linker-equipped D-glucose derivative as the hapten. A high degree of incorporation of the latter into CSA was achieved with high efficiency, and the use of a large excess of the ligand was not necessary. Conjugation of the immunodominant monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, bearing the same spacer, followed a similar pattern, showing that the nature of the carbohydrate does not substantially affect the outcome of the conjugation and that a predicted degree of antigen-loading onto carrier protein is possible to achieve.

Nodding syndrome may be an autoimmune reaction to the parasitic worm Onchocerca volvulus

Patients with nodding syndrome have autoantibodies to leiomodin-1 that are neurotoxic and cross-react with proteins of the parasitic worm Onchocerca volvulus. Linking parasitic infection to autoimmune epilepsy Nodding syndrome is a unique seizure disorder affecting children in parts of East Africa. The cause of nodding syndrome has been an enigma, although an epidemiological association with the parasite Onchocerca volvulus has been established. Johnson et al. demonstrate that patients with nodding syndrome have autoantibodies to leiomodin-1 that are neurotoxic in vitro and that leiomodin-1 is expressed in regions of the brain affected during disease. Leiomodin-1 antibodies cross-react with O. volvulus proteins, linking the parasite to the autoantibody. Thus, nodding syndrome may be an autoimmune epilepsy initiated by a parasitic infection and may be preventable by treatment with antiparasitic strategies such as the drug ivermectin. Nodding syndrome is an epileptic disorder of unknown etiology that occurs in children in East Africa. There is an epidemiological association with Onchocerca volvulus, the parasitic worm that causes onchocerciasis (river blindness), but there is limited evidence that the parasite itself is neuroinvasive. We hypothesized that nodding syndrome may be an autoimmune-mediated disease. Using protein chip methodology, we detected autoantibodies to leiomodin-1 more abundantly in patients with nodding syndrome compared to unaffected controls from the same village. Leiomodin-1 autoantibodies were found in both the sera and cerebrospinal fluid of patients with nodding syndrome. Leiomodin-1 was found to be expressed in mature and developing human neurons in vitro and was localized in mouse brain to the CA3 region of the hippocampus, Purkinje cells in the cerebellum, and cortical neurons, structures that also appear to be affected in patients with nodding syndrome. Antibodies targeting leiomodin-1 were neurotoxic in vitro, and leiomodin-1 antibodies purified from patients with nodding syndrome were cross-reactive with O. volvulus antigens. This study provides initial evidence supporting the hypothesis that nodding syndrome is an autoimmune epileptic disorder caused by molecular mimicry with O. volvulus antigens and suggests that patients may benefit from immunomodulatory therapies.

Gβγ Affinity for Bovine Rhodopsin Is Determined by the Carboxyl-terminal Sequences of the γ Subunit

Two native βγ dimers, β1γ1 and β1γ2, display very different affinities for receptors. Since these γ subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gβγ interaction with receptors. We constructed baculoviruses encoding γ1 and γ2 subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the γ chains and a set of γ1 and γ2 chimeras with the γ2 CAAX motif at the carboxyl terminus. All the γ constructs coexpressed with β1 in Sf9 cells yielded β1γ dimers, which were purified to near homogeneity, and their affinities for receptors and Gα were quantitatively determined. Whereas alteration of the isoprenoid of γ1 from farnesyl to geranylgeranyl and of γ2 from geranylgeranyl to farnesyl had no impact on the affinities of β1γ dimers for Gαt, the non-prenylated β1γ2 dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the β1γ2dimer for rhodopsin and a <3-fold change for the β1γ1 dimer. In each case with identical isoprenylation, the β1γ2 dimer displayed significantly greater affinity for rhodopsin compared with the β1γ1 dimer. Furthermore, dimers containing chimeric Gγ chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Gγ subunit is essential for binding to both Gα and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Gα. Finally, the primary structure of the Gγ subunit provides a major contribution to receptor binding of Gβγ, with the carboxyl-terminal sequence conferring receptor selectivity.

Tracking pathology with proteomics: Identification ofin vivo degradation products of αB-crystallin

Soemmerrings ring is one form of “after cataract” that can occur following cataract surgery. The ring structure is formed by adherence of the anterior lens capsule to the posterior lens capsule. Epithelial cells remaining after surgery differentiate into lens fiber cells but the resulting tissue mass does not remain transparent. The protein in normal lens and in Soemmerrings rings from four individuals was analyzed using two‐dimensional (2‐D) gel electrophoresis, matrix assisted laser desorption/ionization‐time of‐flight‐mass spectrometry (MALDI‐TOF‐MS) and image analysis with Phoretix software. The 2‐D protein patterns of the Soemmerrings rings were generally similar to that of cortical fiber cells of normal human lens with some notable exceptions. Several post‐translationally modified forms of αB‐crystallin(1—175) were identified. Two degradation products, αB‐crystallin(1—170) and αB‐crystallin(1—174), each make up 9.5—27% of the total αB‐crystallin in the Soemmerrings rings and less than 1% in the normal lenses. Other modified forms of αB‐crystallin are aberrant in the fiber cells of the Soemmerring rings relative to normal lens.

A Proteomic and Transcriptomic Approach Reveals New Insight into β-methylthiolation of Escherichia coli Ribosomal Protein S12

β-methylthiolation is a novel post-translational modification mapping to a universally conserved Asp 88 of the bacterial ribosomal protein S12. This S12 specific modification has been identified on orthologs from multiple bacterial species. The origin and functional significance was investigated with both a proteomic strategy to identify candidate S12 interactors and expression microarrays to search for phenotypes that result from targeted gene knockouts of select candidates. Utilizing an endogenous recombinant E. coli S12 protein with an affinity tag as bait, mass spectrometric analysis identified candidate S12 binding partners including RimO (previously shown to be required for this post-translational modification) and YcaO, a conserved protein of unknown function. Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype suggesting that YcaO and RimO likely share a common function. As a follow up, quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12. Collectively, these results indicate that the YcaO protein is involved in β-methylthiolation of S12 and its absence impairs the ability of RimO to modify S12. Additionally, the proteomic data from this study provides direct evidence that the E. coli specific β-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit.

Analysis of TRPC3-interacting proteins by tandem mass spectrometry.

Mammalian transient receptor potential canonical (TRPC) channels are a family of nonspecific cation channels that are activated in response to stimulation of phospholipase C (PLC)-dependent hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. Despite extensive studies, the mechanism(s) involved in regulation of mammalian TRPC channels remains unknown. Presence of various protein-interacting domains in TRPC channels have led to the suggestion that they associate with proteins that are involved in their function and regulation. This study was directed toward identifying the proteins associated with native TRPC3 using a shotgun proteomic approach. Anti-TRPC3 antibody was used to immunoprecipitate TRPC3 from solubilized rat brain crude membranes under conditions that allow retention of TRPC3 function. Proteins in the TRPC3 (using anti-TRPC3 antibody) and control (using rabbit IgG) immunoprecipitates were separated by SDS-PAGE, the gel was sectioned, and the resolved proteins were digested by trypsin in situ. After extraction of the peptides, the peptides were separated by HPLC and sequences derived by MS/MS. Analysis of the data revealed 64 specific TRPC3-associated proteins which can be grouped in terms of their cellular location and involvement in specific cellular function. Many of the proteins identified have been previously reported as TRPC3-regulatory proteins, such as IP3Rs and vesicle trafficking proteins. In addition, we report novel putative TRPC3-interacting proteins, including those involved in protein endocytosis and neuronal growth. To our knowledge, this is the first comprehensive proteomic analysis of a native TRPC channel. These data reveal potential TRPC3 regulatory proteins and provide novel insights of the mechanism(s) regulating TRPC3 channels as well as the possible cellular functions where the channel might be involved.

Linking carbohydrates to proteins using N-(2,2-dimethoxyethyl)-6-hydroxy hexanamide

Abstract The title dimethyl acetal 4 and related compounds can be efficiently synthesized by treatment of 6-caprolactone with commercially available dialkyl acetals. Conventional glucosylation using 4 as a glycosyl acceptor gave mainly β-glycosides which were deprotected to give 13 . The latter was converted to the corresponding aldehyde 16 , which was used as a hapten in conjugation, by reductive amination, to chicken serum albumin (CSA). Effect of reaction time, concentration and molar ratio of hapten 16 to the number of L-lysine residue in CSA upon incorporation of the hapten was studied using MALDI-TOF spectrometry. High loading of CSA with hapten, up to 22 moles of 16 /CSA, could be achieved with 32% efficiency of utilization of the hapten. A glycoconjugate from a derivative, analogous to 16 , of the monosaccharide determinant of the O-PS of Vibrio cholerae O:1, serotype Ogawa and CSA was also prepared. The achieved incorporation of the Vibrio cholerae hapten was in remarkable agreement with the value expected, based on the study with the D-glucose derivative.