Kyoko Kojima

Abundant Lipid and Protein Components of Drusen

Background Drusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death. Methodology/Principal Findings Druse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)2 analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5±13.7, 36.9±12.9, and 43.0±11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37–44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit β, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence. Conclusions/Significance At least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.

Inactivation of Smad4 Accelerates KrasG12D-Mediated Pancreatic Neoplasia

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of <5%. Genetic analysis of PDAC patient samples has shown that specific disease-associated mutations are correlated with histologically defined stages of neoplastic progression in the ductal epithelium. Activating mutations in KRAS are almost uniformly present in early-stage disease, with subsequent inactivating mutations in p16(INK4A), p53, and SMAD4 occurring in more advanced lesions. In this study, we have tested whether the loss of Smad4 would cooperate with an activating Kras(G12D) mutation to promote progression to PDAC using the Pdx1-Cre transgenic system to activate Kras(G12D) and delete Smad4 in all pancreatic lineages including the ductal epithelium. Analysis of double-mutant mice showed that loss of Smad4 significantly accelerated the progression of pancreatic intraepithelial neoplasias (mPanIN) and promoted a high incidence of intraductal papillary mucinous neoplasia and active fibrosis compared with Pdx1-Cre;Kras(G12D) or Pdx1-Cre;Smad4(lox/lox) mice. Occasionally, double-mutant mice progressed to locally invasive PDAC with little evidence of metastases by 6 months of age and without the detectable loss of p53 or p16(Ink4A) expression or function. The loss of Smad4 only seemed to promote disease progression in the presence of the activated Kras(G12D) allele because we observed no abnormal pathology within the pancreata of 23 Pdx1-Cre;Smad4(lox/lox) animals that were analyzed up to 8 months of age. This indicates that Smad4 is dispensable for normal pancreatic development but is critical for at least partial suppression of multiple Kras(G12D)-dependent disease-associated phenotypes.

LRRK2 secretion in exosomes is regulated by 14-3-3

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinsons disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials.

Applying Proteomic-Based Biomarker Tools for the Accurate Diagnosis of Pancreatic Cancer

BackgroundThe proteome varies with physiologic and disease states. Few studies have been reported that differentiate the proteome of those with pancreatic cancer.AimTo apply proteomic-based technologies to body fluids. To differentiate pancreatic neoplasia from nonneoplastic pancreatic disease.MethodsSamples from 50 patients (15 healthy (H), 24 cancer (Ca), 11 chronic pancreatitis (CP)) were prospectively collected and underwent analysis. A high-throughput method, using high-affinity solid lipophilic extraction resins, enriched low molecular weight proteins for extraction with a high-speed 200-Hz matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-MS; Bruker Ultraflex III). Samples underwent software processing with FlexAnalysis, Clinprot, MatLab, and Statistica (baseline, align, and normalize spectra). Nonparametric pairwise statistics, multidimensional scaling, hierarchical analysis, and leave-one-out cross validation completed the analysis. Sensitivity (sn) and specificity (sp) of group comparisons were determined. Two top-down-directed protein identification approaches were combined with MALDI-MS and tandem mass spectrometry to fully characterize the most significant protein biomarker.ResultsUsing eight serum features, we differentiated Ca from H (sn 88%, sp 93%), Ca from CP (sn 88%, sp 30%), and Ca from both H and CP combined (sn 88%, sp 66%). In addition, nine features obtained from urine differentiated Ca from both H and CP combined with high efficiency (sn 90%, sp 90%). Interestingly, the plasma samples (considered by the Human Proteome Organization to be the preferred biological fluid) did not show significant differences. Multidimensional scaling indicated that markers from both serum and urine led to a highly effective clinical indicator of each specific disease state.ConclusionsThe proteomic analysis of noninvasively acquired biological fluids provided a high level of predictability for diagnosing pancreatic cancer. While the proteomic analysis of serum was capable of screening individuals for pancreatic disease (i.e., CP and Ca vs. H), specific urine biomarkers further distinguished malignancy (Ca) from chronic inflammation (CP).

Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human

A number of reports have recently emerged with focus on extraction of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D‐nanoLC‐MS(MS)2 following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.

Alterations in the rat serum proteome induced by prepubertal exposure to bisphenol a and genistein.

Humans are exposed to an array of chemicals via the food, drink and air, including a significant number that can mimic endogenous hormones. One such chemical is Bisphenol A (BPA), a synthetic chemical that has been shown to cause developmental alterations and to predispose for mammary cancer in rodent models. In contrast, the phytochemical genistein has been reported to suppress chemically induced mammary cancer in rodents, and Asians ingesting a diet high in soy containing genistein have lower incidence of breast and prostate cancers. In this study, we sought to: (1) identify protein biomarkers of susceptibility from blood sera of rats exposed prepubertally to BPA or genistein using Isobaric Tandem Mass Tags quantitative mass spectrometry (TMT-MS) combined with MudPIT technology and, (2) explore the relevance of these proteins to carcinogenesis. Prepubertal exposures to BPA and genistein resulted in altered expression of 63 and 28 proteins in rat sera at postnatal day (PND) 21, and of 9 and 18 proteins in sera at PND35, respectively. This study demonstrates the value of using quantitative proteomic techniques to explore the effect of chemical exposure on the rat serum proteome and its potential for unraveling cellular targets altered by BPA and genistein involved in carcinogenesis.

Comparative proteomics analysis of high n-butanol producing metabolically engineered Clostridium tyrobutyricum

The acidogenic Clostridium tyrobutyricum has recently been metabolically engineered to produce n-butanol. The objective of this study was to obtain a comprehensive understanding as to how butanol production was regulated in C. tyrobutyricum to guide the engineering of next-generation strains. We performed a comparative proteomics analysis, covering 78.1% of open reading frames and 95% of core enzymes, using wild type, ACKKO mutant (Δack) producing 37.30 g/L of butyrate and ACKKO-adhE2 mutant (Δack-adhE2) producing 16.68 g/L of butanol. In ACKKO-adhE2, the expression of most glycolytic enzymes was decreased, the thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) that convert acetyl-CoA to butyryl-CoA were increased, and the heterologous bifunctional acetaldehyde/alcohol dehydrogenase (adhE2) catalyzing butanol formation was highly expressed. The apparent imbalance of energy and redox was observed due to the downregulation of acids production and the addition of butanol synthesis pathway, which also resulted in increased expression of chaperone proteins and glycerol-3-phosphate dehydrogenase (glpA) and the silence of sporulation transcription factor Spo0A (spo0A) as the cellular responses to butanol production. This study revealed the mechanism of carbon redistribution, and limiting factors and rational metabolic cell and process engineering strategies to achieve high butanol production in C. tyrobutyricum.

Analysis of the Human Proteome in Subcutaneous and Visceral Fat Depots in Diabetic and Non-diabetic Patients with Morbid Obesity

No longer regarded as simply a storage depot, fat is a dynamic organ acting locally and systemically to modulate energy homeostasis, glucose sensitivity, insulin resistance, and inflammatory pathways. Here, mass spectrometry was used to survey the proteome of patient matched subcutaneous fat and visceral fat in 20 diabetic vs 22 nondiabetic patients with morbid obesity. A similar number of proteins (~600) were identified in each tissue type. When stratified by diabetic status, 19 and 41 proteins were found to be differentially abundant in subcutaneous fat and omentum, respectively. These proteins represent pathways known to be involved in metabolism. Five of these proteins were differentially abundant in both fat depots: moesin, 78 kDa glucose-regulated protein, protein cordon-bleu, zinc finger protein 611, and cytochrome c oxidase subunit 6B1. Three proteins, decorin, cytochrome c oxidase subunit 6B1, and 78 kDa glucose-regulated protein, were further tested for validation by western blot analysis. Investigation of the proteins reported here is expected to expand on the current knowledge of adipose tissue driven biochemistry in diabetes and obesity, with the ultimate goal of identifying clinical targets for the development of novel therapeutic interventions in the treatment of type 2 diabetes mellitus. To our knowledge, this study is the first to survey the global proteome derived from each subcutaneous and visceral adipose tissue obtained from the same patient in the clinical setting of morbid obesity, with and without diabetes. It is also the largest study of diabetic vs nondiabetic patients with 42 patients surveyed.

Surveying the serologic proteome in a tissue-specific kras(G12D) knockin mouse model of pancreatic cancer

We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue‐specific KrasG12D‐knockin mouse model of pancreatic cancer that consistently forms precancerous lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top‐associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN‐γ, VEGF and GM‐CSF were significantly increased in serum from the KrasG12D animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early‐stage neoplasia.

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Background: Tdp1 repair of adducted DNA involves a covalent enzyme-DNA intermediate, formed and resolved by Hisnuc and Hisgab residues. Stabilized Hisgab mutant-DNA complexes are cytotoxic. Results: DNA adduct cleavage by an adjacent His residue in HisnucAla mutants impair enzyme-DNA intermediate resolution. Conclusion: Alterations in active site geometry enhance the stability of cytotoxic Tdp1-DNA intermediates. Significance: These findings provide the rationale for developing chemotherapeutics that poison Tdp1. Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.