A human protein atlas for normal and cancer tissues based on antibody proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

Discovery and Validation of New Potential Biomarkers for Early Detection of Colon Cancer

Background

Accurate detection of characteristic proteins secreted by colon cancer tumor cells in biological fluids could serve as a biomarker for the disease. The aim of the present study was to identify and validate new serum biomarkers and demonstrate their potential usefulness for early diagnosis of colon cancer.

Methods

The study was organized in three sequential phases: 1) biomarker discovery, 2) technical and biological validation, and 3) proof of concept to test the potential clinical use of selected biomarkers. A prioritized subset of the differentially-expressed genes between tissue types (50 colon mucosa from cancer-free individuals and 100 normal-tumor pairs from colon cancer patients) was validated and further tested in a series of serum samples from 80 colon cancer cases, 23 patients with adenoma and 77 cancer-free controls.

Results

In the discovery phase, 505 unique candidate biomarkers were identified, with highly significant results and high capacity to discriminate between the different tissue types. After a subsequent prioritization, all tested genes (N = 23) were successfully validated in tissue, and one of them, COL10A1, showed relevant differences in serum protein levels between controls, patients with adenoma (p = 0.0083) and colon cancer cases (p = 3.2e-6).

Conclusion

We present a sequential process for the identification and further validation of biomarkers for early detection of colon cancer that identifies COL10A1 protein levels in serum as a potential diagnostic candidate to detect both adenoma lesions and tumor.

Impact

The use of a cheap serum test for colon cancer screening should improve its participation rates and contribute to decrease the burden of this disease.     

Analysis of Alpha-Synuclein in Malignant Melanoma – Development of a SRM Quantification Assay

Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.     

Cholesterol impairs autophagy-mediated clearance of amyloid beta while promoting its secretion

Macroautophagy/autophagy failure with the accumulation of autophagosomes is an early neuropathological feature of Alzheimer disease (AD) that directly affects amyloid beta (Aβ) metabolism. Although loss of presenilin 1 function has been reported to impair lysosomal function and prevent autophagy flux, the detailed mechanism leading to autophagy dysfunction in AD remains to be elucidated. The resemblance between pathological hallmarks of AD and Niemann-Pick Type C disease, including endosome-lysosome abnormalities and impaired autophagy, suggests cholesterol accumulation as a common link. Using a mouse model of AD (APP-PSEN1-SREBF2 mice), expressing chimeric mouse-human amyloid precursor protein with the familial Alzheimer Swedish mutation (APP695swe) and mutant presenilin 1 (PSEN1-dE9), together with a dominant-positive, truncated and active form of SREBF2/SREBP2 (sterol regulatory element binding factor 2), we demonstrated that high brain cholesterol enhanced autophagosome formation, but disrupted its fusion with endosomal-lysosomal vesicles. The combination of these alterations resulted in impaired degradation of Aβ and endogenous MAPT (microtubule associated protein tau), and stimulated autophagy-dependent Aβ secretion. Exacerbated Aβ-induced oxidative stress in APP-PSEN1-SREBF2 mice, due to cholesterol-mediated depletion of mitochondrial glutathione/mGSH, is critical for autophagy induction. In agreement, in vivo mitochondrial GSH recovery with GSH ethyl ester, inhibited autophagosome synthesis by preventing the oxidative inhibition of ATG4B deconjugation activity exerted by Aβ. Moreover, cholesterol-enrichment within the endosomes-lysosomes modified the levels and membrane distribution of RAB7A and SNAP receptors (SNAREs), which affected its fusogenic ability. Accordingly, in vivo treatment with 2-hydroxypropyl-β-cyclodextrin completely rescued these alterations, making it a potential therapeutic tool for AD.     

Clumping factor A-mediated virulence during Staphylococcus aureus infection is retained despite fibrinogen depletion

Clumping factor A (ClfA), a fibrinogen-binding protein expressed on the Staphylococcus aureus cell surface, has previously been shown to act as a virulence factor in experimental septic arthritis. Although the interaction between ClfA and fibrinogen is assumed to be of importance for the virulence of S. aureus, this has not been demonstrated in any in vivo model of infection. Therefore, the objective of this study was to investigate the contribution of this interaction to ClfA-mediated virulence in murine S. aureus-induced arthritis. Ancrod, a serine protease with thrombin-like activity, was used to induce in vivo depletion of fibrinogen in mice. Ancrod treatment significantly aggravated septic arthritis following inoculation with a ClfA-expressing strain (Newman) compared to control treatment. Also, ancrod treatment tended to enhance the arthritis induced by a clfA mutant strain (DU5876), indicating that fibrinogen depletion exacerbates septic arthritis in a ClfA-independent manner. Most importantly, the ClfA-expressing strain was much more arthritogenic than the isogenic clfA mutant, following inoculation of fibrinogen-depleted mice. This finding indicates that the interaction between ClfA and free fibrinogen is not required for ClfA-mediated functions contributing to S. aureus virulence. It is conceivable that ClfA contributes to the virulence of S. aureus through interactions with other host ligands than fibrinogen.     

Stereopsidales - A New Order of Mushroom-Forming Fungi

One new order, one new family, and one new combination are presented, as the result of molecular phylogenetic analyses. The new order Stereopsidales and the new family Stereopsidaceae are described incorporating Stereopsis radicans and S. globosa, formerly Clavulicium globosum. We show that not only do these species represent an old overlooked lineage, but both species harbor cryptic diversity. In addition, a third species, C. macounii, appears as a plausible sister to the new lineage, but there is conflict in the data. All specimens of S. radicans and S. globosa analysed here are from the South and Central Americas; several records of S. radicans have been made also from tropical Asia. We expect the true diversity in this group to be a lot higher than presented in this paper. Stereopsis radicans was formerly included in Polyporales, but a placement within that order is rejected by our data through SH tests. The dataset consisted of four nuclear markers: rpb2, tef1, LSU and SSU, each of which was analysed separately using maximum likelihood and Bayesian inference. Recombination detection tests indicate no plausible recombinations. The potential of S. radicans, S. globosa and C. macounii being amphitallic is briefly discussed.     

THE COLONIZATION OF DROSOPHILA SUBOBSCURA IN CHILE. II. CLINES IN THE CHROMOSOMAL ARRANGEMENTS

Drosophila subobscura is a Palearctic species that was first detected in the New World in Puerto Montt (Chile) in February 1978. Since that time, it has spread over a broad area and increased in population density. The South American populations exhibit a high level of chromosomal polymorphism: 20 different arrangements exist, distributed among five chromosomes. Chromosomal arrangement heterozygosity varies from 0.55 to 0.61 in the nine populations examined. Incipient clines in the frequencies of the arrangements are appearing; these clines follow the same latitudinal direction as in the Old World. Wing length significantly decreases with latitude, as it does in Europe. The colonization of South America by D. subobscura appears to be a major natural experiment with outcomes that duplicate the distributional patterns—in chromosomal polymorphism and in wing length—observed in the Old World, thereby strongly supporting the adaptive significance of these patterns. The data show a very rapid effect of natural selection promoting genetic differentiation among natural populations.