In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2-3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the in vitro aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37°C undergo a random coil to ␤-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, ␤-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal͞mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.

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The repeat length-dependent tendency of the polyglutamine sequences of certain proteins to form aggregates may underlie the cytotoxicity of these sequences in expanded CAG repeat diseases such as Huntington's disease. We report here a number of features of various polyglutamine (polyGln) aggregates and their assembly pathways that bear a resemblance to generally recognized defining features of amyloid fibrils. PolyGln aggregation kinetics displays concentration and length dependence and a lag phase that can be abbreviated by seeding. PolyGln aggregates exhibit classical beta-sheet-rich circular dichroism spectra consistent with an amyloid-like substructure. The fundamental structural unit of all the in vitro aggregates described here is a filament about 3 nm in width, resembling the protofibrillar intermediates in amyloid fibril assembly. We observed these filamentous structures either as isolated threads, as components of ribbonlike sheets, or, rarely, in amyloid-like twisted fibrils. All of the polyGln aggregates described here bind thioflavin T and shift its fluorescence spectrum. Although all polyGln aggregates tested bind the dye Congo red, only aggregates of a relatively long polyGln peptide exhibit Congo red birefringence, and this birefringence is only observed in a small portion of these aggregates. Remarkably, a monoclonal antibody with high selectivity for a generic amyloid fibril conformational epitope is capable of binding polyGln aggregates. Thus, polyGln aggregates exhibit most of the characteristic features of amyloid, but the twisted fibril structure with Congo red birefringence is not the predominant form in the polyGln repeat length range studied here. We also find that polyGln peptides exhibit an unusual freezing-dependent aggregation that appears to be caused by the freeze concentration of peptide and/or buffer components. This is of both fundamental and practical significance. PolyGln aggregation is revealed to be a highly specific process consistent with a significant degree of order in the molecular structure of the product. This ordered structure, or the assembly process leading to it, may be responsible for the cell-specific neuronal degeneration observed in Huntington's and other expanded CAG repeat diseases.

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Carbohydrate post-translational modifications on proteins are important determinants of protein function in both normal and disease biology. We have developed a method to allow the efficient, multiplexed study of glycans on individual proteins from complex mixtures, using antibody microarray capture of multiple proteins followed by detection with lectins or glycan-binding antibodies. Chemical derivatization of the glycans on the spotted antibodies prevented lectin binding to those glycans. Multiple lectins could be used as detection probes, each targeting different glycan groups, to build up lectin binding profiles of captured proteins. By profiling both protein and glycan variation in multiple samples using parallel sandwich and glycan-detection assays, we found cancer-associated glycan alteration on the proteins MUC1 and CEA in the serum of pancreatic cancer patients. Antibody arrays for glycan detection are highly effective for profiling variation in specific glycans on multiple proteins and should be useful in diverse areas of glycobiology research.

A method is described for dissolving and disaggregating chemically synthesized polyglutamine peptides. Polyglutamine peptides longer than about Q 20 have been reported to be insoluble in water, but dissolution in -and evaporation from -a mixture of trifluoroacetic acid and hexafluoroisopropanol converts polyglutamine peptides up to at least Q 44 to a form readily soluble in aqueous buffers. This procedure also has a dramatic effect on peptides which appear to be completely soluble in water, by removing traces of aggregate that seed aggregation. The protocol makes possible solution studies-including in vitro aggregation experiments-on polyglutamine peptides with repeat lengths associated with increased risk of Huntington's Disease and other expanded CAG repeat diseases. It may also be useful in conducting reproducible, quantitative aggregation studies on other polypeptides.Keywords: Polyglutamine; disaggregation; nucleation-dependent aggregation; seed; Huntington's diseaseThe misassembly of proteins and peptides into highly insoluble, noncovalent aggregates is a process of tremendous practical and fundamental importance. The formation of amyloid fibrils and other aggregates in vivo plays a role in a number of human neurodegenerative (Martin 1999) and other (Sipe 1992) diseases, as well as a growing list of non-Mendelian trait transfers in microbial genetics (Lindquist 1997). Aggregate formation is also important in biotechnology, controlling both inclusion body formation in recombinant expression (Mukhopadhyay 1997) and the efficiency with which dissolved and denatured inclusion bodies refold (Bernardez-Clark et al. 1999). The existence of the molecular chaperones (Fink and Goto 1998), a family of proteins responsible for suppressing aggregate formation in the cell, suggests that the efficiency of cellular protein biosynthesis is normally tempered by the intrinsic propensity of polypeptides to enter aggregation side reactions during the folding process. Many of these aggregation processes can be effectively modeled in simple, defined systems in vitro, making possible important studies on the mechanisms of assembly and the structures of the assembled products (Kelly and Lansbury 1994). In some cases, however, the apparent insolubility of the starting peptide in denaturing solvents places significant limitations on our ability to study fundamental aspects of aggregation. In other cases, studies of an apparently soluble peptide by different laboratories give dramatically different aggregation kinetics consistent with the presence of variable trace amounts of aggregation seeds in the starting materials.The genetic defect in the expanded CAG repeat diseases involves a length increase of normally benign polyglutamine (polyGln) sequences in certain proteins, generally from a wild-type length <38 Gln residues to a pathological length greater than about 40 Gln residues (Cummings and Zoghbi 2000). Because one hypothesis of the disease mechanism is that polyGln aggregation is responsible for neurotoxicity (Cummings ...

Purpose CA-125 has been a valuable marker for detecting ovarian cancer, however, not sensitive enough to detect early stage disease and not specific for ovarian cancer. The purpose of our study was to identify autoantibody markers that are specific for ovarian cancer regardless of CA-125 levels. Methods Top-down and iTRAQ quantitative proteomics methods were used to identify high frequency autoantibodies in ovarian cancer. Protein microarrays comprising the recombinant autoantigens were screened using serum samples from various stages of ovarian cancer with diverse levels of CA-125 as well as benign and healthy controls. ROC curve and dot blot analyses were performed to validate the sensitivity and specificity of the autoantibody markers. Results The proteomics methodologies identified >60 potential high frequency autoantibodies in ovarian cancer. Individual serum samples from ovarian cancer stages I-IV compared to control samples that were screened on a microarray containing native recombinant autoantigens revealed a panel of stage I high frequency autoantibodies. Preliminary ROC curve and dot blot analyses performed with the ovarian cancer samples showed higher specificity and sensitivity as compared to CA-125. Three autoantibody markers exhibited higher specificity in various stages of ovarian cancer with low and normal CA-125 levels. Conclusions Proteomics technologies are suitable for the identification of protein biomarkers and also the identification of autoantibody biomarkers when combined with protein microarray screening. Using native recombinant autoantigen arrays to screen autoantibody markers, it is possible to identify markers with higher sensitivity and specificity than CA-125 that are relevant for early detection of ovarian cancer.

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