Revealing the cellular degradome by mass spectrometry analysis of proteasome-cleaved peptides.

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals. Further, we performed analysis of minute amounts of clinical samples by studying cells from the peripheral blood of patients with systemic lupus erythematosus (SLE). We found increased degradation of histones in patient immune cells, thereby suggesting a role of aberrant proteasomal degradation in the pathophysiology of SLE. Thus, MAPP offers a broadly applicable method to facilitate the study of the cellular-degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

Post-Translational Modification Profiling-Functional Proteomics for the Analysis of Immune Regulation.

Posttranslational modifications (PTMs) of proteins are an integral part of major cellular regulatory mechanisms dictating protein function, localization, and stability. The capacity to screen PTMs using protein microarrays has advanced our ability to identify their targets and regulatory role. This chapter discusses a unique procedure that combines functional extract-based activity assay with large-scale screening utilities of protein microarrays. This "PTM-profiling" system offers advantages in quantitatively identifying modifications in an unbiased manner in the context of specific cellular conditions. While the possibilities of studying PTMs in different settings are enormous, the immune system presents an attractive model for studying the effects of perturbations in PTMs, and specifically the ubiquitin system, as these were already implicated in both immune function and dysfunction. This chapter discusses the significance of PTM profiling in addressing basic questions in immunology. We describe detailed protocols for the preparation of functional cell extracts from immune cell cultures, following differentiation or induced signals, and screening PTMs on protein arrays, as well as basic guidelines for data analysis and interpretation.

Core promoter sequence in yeast is a major determinant of expression level.

The core promoter is the regulatory sequence to which RNA polymerase is recruited and where it acts to initiate transcription. Here, we present the first comprehensive study of yeast core promoters, providing massively parallel measurements of core promoter activity and of TSS locations and relative usage for thousands of native and designed sequences. We found core promoter activity to be highly correlated to the activity of the entire promoter and that sequence variation in different core promoter regions substantially tunes its activity in a predictable way. We also show that location, orientation, and flanking bases critically affect TATA element function, that transcription initiation in highly active core promoters is focused within a narrow region, that poly(dA:dT) orientation has a functional consequence at the 3' end of promoters, and that orthologous core promoters across yeast species have conserved activities. Our results demonstrate the importance of core promoters in the quantitative study of gene regulation.

Systematic dissection of the sequence determinants of gene 3' end mediated expression control.

The 3'end genomic region encodes a wide range of regulatory process including mRNA stability, 3' end processing and translation. Here, we systematically investigate the sequence determinants of 3' end mediated expression control by measuring the effect of 13,000 designed 3' end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3' end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3' end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3' UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3'UTRs, confirming the role for this element in 3' end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3' end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3' end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3' end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.