Abstract (provided by applicant):

Proteolysis is of paramount importance to biological regulation, and its significance is magnified because it is irreversible. Proteolysis regulates the four fundamental aspects of cell behavior: division, death, differentiation and motility. Proteolytic pathways are key to the pathology of virtually every type of human disease including, infection, inflammation, thrombosis, cancer, emphysema, Alzheimer's, etc. The importance and impact of understanding proteolysis "in total" will be immense. Achieving this ultimate goal will be a considerable challenge, and one that will require an ambitious and coordinated effort. Here we propose to form the Center on Proteolytic Pathways (CPP), a National Resource for the study of proteases, their inhibitors, their products, and upstream and downstream regulatory pathways. The strategic goal of the CPP is to develop The Protease Pathway Interrogation Platform (PPIP) technology. This platform technology will be comprised of four foundation technologies: 1.) Activity-based Protease Profiling; 2.) Protease Activity Imaging Technology; 3.) Product Terminal Isotope Coding; and 4.) the in silico environment called Protease Map. A key and unique feature of this technology platform is its focus squarely on measuring activity, as opposed to just abundance. The CPP will contain a Training component to ensure that a new cadre of scientists is trained to tackle protease biology with the most modem of techniques and most cutting-edge technology. An Outreach component is planned to ensure that the CPP achieves high visibility, and ultimately numerous connections with the scientific research community.

Abstract (provided by applicant):

Protein modification on histone lysines is critical for controlling gene expression, which itself controls the incredibly variable and plastic expression of the proteome in diverse cell types. Modifications on lysine are chemically diverse and include acetylation, methylation, ubiquitylation, and sumoylation. Increasingly, acetyl- and methyl-lysines are being discovered in a host of other proteins, only some of which directly control gene expression. Ubiquitylation controls the life and death of most proteins, as well as many other diverse protein functions. Remarkably, the pathways regulating the interplay between these diverse modifications on lysines have been very little studied. The network of these modification pathways is very poorly understood indeed; many lysine-modifying proteins are encoded by multi-gene families, with redundant activities, and have multiple substrates. Thus genetic and computational approaches for decrypting such redundancy are needed to dissect the complex networks defined by these signaling pathways. This proposal combines such techniques with a new and innovative proteomics technology, protein microarrays, and a new affinity ligand technology for identifying novel acetyltransferases. These newer approaches will be complemented in this Technology Center for Networks and Pathways by a heavy emphasis on the development and application of innovative mass spectrometry technologies, including sensitive technologies for quantifying dynamics of lysine modification in cells. Dissecting how lysine modifications change in response to biological stimuli is in its infancy largely because sensitive and robust experimental techniques for quantifying protein modification are needed and will be developed here. A unique instrumentation system aimed at profiling lysine modification in single cells will be developed, which will open a whole new world of single-cell profiling to examine the epigenetic changes that occur in individual cells as they develop, as they age, or as cancer progresses. Diverse Driving Biological Projects centered on lysine acetylation, methylation, and ubiquitylation, as well as Training and Technology Dissemination efforts, are extensively integrated with the Technology Development components of the proposal. Because lysine modification is intertwined with human health, aging, and disease at many levels, these technologies could have far-reaching effects.