Alpha-synuclein (αSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons αSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following αSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic αSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against αSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.Parkinson's disease (PD) is the second most common neurodegenerative disorder (1,2). Accruing evidence points to a causative role for the presynaptic protein alpha-synuclein (αSyn) in PD pathogenesis. αSyn is a major constituent of Lewy Bodies-cellular inclusions that are the hallmark pathological feature of PD and other neurodegenerative disorders collectively

The association of cdk4 with D-type cyclins to form functional kinase complexes is comparatively inefficient. This has led to the suggestion that assembly might be a regulated step. In this report we demonstrate that the CDK inhibitors pZl'^^'', p27^^^, and p57^^^^ all promote the association of cdk4 with the D-type cyclins. This effect is specific and does not occur with other cdk inhibitors or cdk-binding proteins. Both in vivo and in vitro, the abundance of assembled cdk4/cyclin D complex increases directly with increasing inhibitor levels. The promotion of assembly is not attributable to a simple cell cycle block and requires the function of both the cdk and cyclin-binding domains. Kinetic studies demonstrate that p21 and p27 lead to a 35-and 80-fold increase in K^, respectively, mostly because of a decrease in X^ff. At low concentrations, p21 promotes the assembly of active kinase complexes, whereas at higher concentrations, it inhibits activity. Moreover, immunodepletion experiments demonstrate that most of the active cdk4-associated kinase activity also associates with p21. To confirm these results in a natural setting, we examine the assembly of endogenous complexes in mammary epithelial cells after release from a GQ arrest. In agreement with our other data, cyclin Dl and p21 bind concomitantly to cdk4 during the in vivo assembly of cdk4/cyclin Dl complexes. This complex assembly occurs in parallel to an increase in cyclin Dl-associated kinase activity. Immunodepletion experiments demonstrate that most of the cellular cyclin Dl-associated kinase activity is also p21 associated. Finally, we find that all three CIP/KIP inhibitors target cdk4 and cyclin Dl to the nucleus. We suggest that in addition to their roles as inhibitors, the p21 family of proteins, originally identified as inhibitors, may also have roles as adaptor proteins that assemble and program kinase complexes for specific functions.

High proportions of autistic children suffer from gastrointestinal (GI) disorders, implying a link between autism and abnormalities in gut microbial functions. Increasing evidence from recent high-throughput sequencing analyses indicates that disturbances in composition and diversity of gut microbiome are associated with various disease conditions. However, microbiome-level studies on autism are limited and mostly focused on pathogenic bacteria. Therefore, here we aimed to define systemic changes in gut microbiome associated with autism and autism-related GI problems. We recruited 20 neurotypical and 20 autistic children accompanied by a survey of both autistic severity and GI symptoms. By pyrosequencing the V2/V3 regions in bacterial 16S rDNA from fecal DNA samples, we compared gut microbiomes of GI symptom-free neurotypical children with those of autistic children mostly presenting GI symptoms. Unexpectedly, the presence of autistic symptoms, rather than the severity of GI symptoms, was associated with less diverse gut microbiomes. Further, rigorous statistical tests with multiple testing corrections showed significantly lower abundances of the genera Prevotella, Coprococcus, and unclassified Veillonellaceae in autistic samples. These are intriguingly versatile carbohydrate-degrading and/or fermenting bacteria, suggesting a potential influence of unusual diet patterns observed in autistic children. However, multivariate analyses showed that autism-related changes in both overall diversity and individual genus abundances were correlated with the presence of autistic symptoms but not with their diet patterns. Taken together, autism and accompanying GI symptoms were characterized by distinct and less diverse gut microbial compositions with lower levels of Prevotella, Coprococcus, and unclassified Veillonellaceae.

Transcription factors (TFs) regulate the expression of genes through sequence-specific interactions with DNA-binding sites. However, despite recent progress in identifying in vivo TF binding sites by microarray readout of chromatin immunoprecipitation (ChIP-chip), nearly half of all known yeast TFs are of unknown DNA-binding specificities, and many additional predicted TFs remain uncharacterized. To address these gaps in our knowledge of yeast TFs and their cis regulatory sequences, we have determined high-resolution binding profiles for 89 known and predicted yeast TFs, over more than 2.3 million gapped and ungapped 8-bp sequences (''k-mers''). We report 50 new or significantly different direct DNA-binding site motifs for yeast DNA-binding proteins and motifs for eight proteins for which only a consensus sequence was previously known; in total, this corresponds to over a 50% increase in the number of yeast DNA-binding proteins with experimentally determined DNA-binding specificities. Among other novel regulators, we discovered proteins that bind the PAC (Polymerase A and C) motif (GATGAG) and regulate ribosomal RNA (rRNA) transcription and processing, core cellular processes that are constituent to ribosome biogenesis. In contrast to earlier data types, these comprehensive k-mer binding data permit us to consider the regulatory potential of genomic sequence at the individual word level. These k-mer data allowed us to reannotate in vivo TF binding targets as direct or indirect and to examine TFs' potential effects on gene expression in ;1700 environmental and cellular conditions. These approaches could be adapted to identify TFs and cis regulatory elements in higher eukaryotes.

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

We report a high-density self assembling protein microarray that displays thousands of proteins, produced and captured in situ from immobilized cDNA templates. Over 1500 unique cDNAs were tested with > 90% success with nearly all proteins displaying yields within 2 fold of the mean, minimal sample variation and good day to day reproducibility. The displayed proteins revealed selective protein interactions. This method will enable various experimental approaches to study protein function in high throughput.

The release of proteins from tumors triggers an immune response in cancer patients. These tumor antigens arise from several mechanisms including tumor-specific alterations in protein expression, mutation, folding, degradation, or intracellular localization. Responses to most tumor antigens are rarely observed in healthy individuals, making the response itself a biomarker that betrays the presence of underlying cancer. Antibody immune responses show promise as clinical biomarkers because antibodies have long half lives in serum, are easy to measure, and are stable in blood samples. However, our understanding of the specificity and the impact of the immune response in early stages of cancer is limited. The immune response to cancer, whether endogenous or driven by vaccines, involves highly specific T lymphocytes (which target tumor-derived peptides bound to self-MHC proteins) and B lymphocytes (which generate antibodies to tumor-derived proteins). T cell target antigens have been identified either by expression cloning from tumor cDNA libraries, or by prediction based on patterns of antigen expression ("reverse immunology"). B cell targets have been similarly been identified using the antibodies in patient sera to screen cDNA libraries derived from tumor cell lines. This review focuses on the application of recent advances in proteomics for the identification of tumor antigens. These advances are opening the door for targeted vaccine development, and for using immune response signatures as biomarkers for cancer diagnosis and monitoring.

MCF10A mammary epithelial cells form growth-arrested structures when cultured in three-dimensional basement membrane gels. Activation of the receptor tyrosine kinase ErbB2 induces formation of proliferative structures that share properties with noninvasive early stage lesions. We conducted a genetic screen to identify cDNAs that can cooperate with ErbB2 to induce migration in these cells, with the hypothesis that they would represent candidate ''second hits'' in the development of invasive breast carcinomas. We found that expression of transforming growth factor (TGF)␤1 and TGF␤3 in cells expressing activated ErbB2 induces migration in transwell chambers and invasive behavior in both basement membrane cultures and invasion chambers. The ability of ErbB2 to cooperate with TGF␤ correlated with sustained, elevated activation of extracellular signal-regulated kinase (Erk)-mitogen-activated protein kinase. Pharmacological reduction of Erk activity inhibited the cooperative effect of TGF␤ and ErbB2 on migration and expression of activated Erk kinase was sufficient to cooperate with TGF␤ to induce migration and invasion, suggesting that sustained Erk activation is critical for ErbB2͞TGF␤ cooperation. In addition, we show that costimulation of ErbB2 and TGF␤ induces autocrine secretion of factors that are sufficient to induce migration, but not invasion, by means of both epidermal growth factor receptor-dependent and -independent processes. These results support the role of TGF␤ as a pro-invasion factor in the progression of breast cancers with activated ErbB2 and suggest that activation of the Erk and epidermal growth factor receptor pathways are key in mediating these events. E rbB2 (HER2͞Neu) is overexpressed in 20% to 30% of invasive breast tumors and up to 85% of comedo type ductal carcinoma in situ (DCIS), an early stage in breast cancer (1, 2). The ErbB receptor family has four members: ErbB1 (EGFR͞ HER1), ErbB2, ErbB3, and ErbB4. When these receptors are stimulated with epidermal growth factor (EGF) family ligands, receptor activation occurs by means of both homo-and heterodimerization among the receptor family members (3). However, under conditions where ErbB2 is amplified or overexpressed, activation can occur by ligand-independent homodimerization (4, 5).Previously, we investigated the consequences of activation of ErbB2 homodimers in the context of a nontransformed human mammary epithelial cell line, MCF10A (6, 7). When grown in basement membrane cultures, MCF10A cells form growth arrested three-dimensional structures, termed acini (8). These structures are comprised of a single layer of polarized epithelial cells surrounding a hollow lumen and resemble the mammary acini that form terminal ductal lobular units in the adult breast (7,8).To study the contributions of ErbB receptors in these cells, we generated chimeras of ErbB1 (p75.B1) and ErbB2 (p75.B2), which are inducibly activated by means of homodimerization by using the dimeric FKBP ligand AP1510 (ARIAD Pharmaceuticals, Cambridge, MA; refs. 9 and 10). T...