SBIR-STTR Award

Mammalian cells remove misfolded proteins by degradation mediated by the ubiquitin (Ub) proteasome pathway and autophagy. The first step in the development of neuronal dysfunction or neuronal death is the lack of a response to misfolded proteins. Dysfunction in the Ub proteasome pathway in neurons leads to the accumulation of aggregates and plaques in neurons. Ubiquitylation of proteins not only controls the half-lives of proteins but also acts as a regulatory modification, e.g., histone ubiquitylation. Due to the dynamic nature of Ub conjugation and deconjugation, ubiquitylated proteins are extremely difficult to isolate and study. Identifying a protein with one particular Ub post translational modification out of the pool of modified proteins becomes nearly impossible without selective tools. A further complication is the presence of multiple types of Ub-Ub linkages in polyubiquitin chains. Ubiquitin is attached, via isopeptide bonds, to lysine residues in the target protein. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs, again through the formation of isopeptide bonds between the C-terminus of one Ub and any of seven (7) lysines, or N-terminus of the target Ub. Ub chains with different linkages convey different messages to cells and, hence, determine the ultimate fate of the protein -- degradation, translocation, and oxidative stress response, to name a few. The precise information encoded in the various chain linkages is largely unknown due to a lack of reagents that selectively recognize these linkages. The goal of this proposal is to develop tools that allow the selective identification, quantification, and isolation of proteins modified by mono and polyubiquitin chains containing difficult to study linkages. This will be accomplished using a novel Ub binding microarray that contains all of the known Ub binding motifs from various proteins. In Phase I, we will identify and characterize novel Ub binding domains (UbDs) exhibiting selectivity for monoubiquitin, monoubiquitylated substrates and specific polyubiquitin linkages. Given the fact that dysregulation of the Ub proteasome pathway and autophagy have been strongly implicated as a first step in neurodegenerative diseases, the development of sensitive and novel chain selective tools will lead to early diagnosis of Alzheimer’s and Parkinson’s diseases (phase II).

Public Health Relevance Statement:
Ubiquitylation and de-ubiquitylation are post-translational modifications that affect the function and turnover of many proteins. Dysregulation of protein ubiquitylation is an initial step in the development of neurodegenerative diseases. The study of specific protein ubiquitylation is difficult due to the low abundance and the complexity of polyubiquitin chains. It is proposed here to generate specific tools that will distinguish among the various forms of polyubiquitin that have been understudied due to lack of reagents. The use of these tools will dramatically enhance our understanding of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease as well as other diseases.

Project Terms:
Affect; Affinity; Alzheimer's Disease; amino group; Applications Grants; Architecture; Autoimmune Diseases; Autophagocytosis; Binding; Biochemical; biological adaptation to stress; Biology; biophysical techniques; C-terminal; Cancer Center; carboxylate; Cell Line; Cells; Chemicals; Communicable Diseases; Communities; Complication; Custom; design; Detection; detector; Development; Disease; Disease model; DNA Repair; Doctor of Medicine; Down-Regulation; drug discovery; Drug Targeting; Early Diagnosis; Endocytosis; Engineering; Enzymes; Excision; Exhibits; experience; experimental study; Functional disorder; Glycine; Goals; Grant; Half-Life; Histones; Immune response; improved; Inflammation; Link; Lysine; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; misfolded protein; Modification; Mono-S; multicatalytic endopeptidase complex; N-terminal; Names; Nature; Neurodegenerative Disorders; neuron development; neuron loss; Neuronal Dysfunction; Neurons; novel; Outcome; Oxidative Stress; Parkinson Disease; Pathogenesis; Pathway interactions; Pattern; Phase; Phosphorylation; Play; Polyubiquitin; Post-Translational Protein Processing; professor; protein degradation; protein function; Protein Microchips; protein structure; protein transport; Proteins; Proteomics; Reagent; receptor; receptor recycling; Research; response; Role; screening; Signal Transduction; Specificity; Staining method; Stains; success; Testing; Texas; Therapeutic; Time; tool; Tube; Ubiquitin; ubiquitin mediated proteasome degradation; Ubiquitination; Universities; Western Blotting

Tools are needed to establish the etiology of Alzheimer’s disease (AD), whose signatures appear before the disease presents. Early in AD is ubiquitylation of misfolded proteins; those not degraded by the proteasome can form neurofibrillary tangles such as ?-amyloid or tau protein aggregates. Ubiquitin (Ub)-mediated signaling plays a central role in protein degradation, and a first step in neurodegeneration is a change in ubiquitylation patterns, particularly, that of poly-Ub chains attached to proteins. Development of sensitive methods to detect these ubiquitylation signatures will be a major advance in AD drug discovery and diagnosis. Poly-Ub chains are formed utilizing all seven lysines, as well as the N-terminus of ubiquitin, suggesting that these chains encode much information. Each linkage, including mono-Ub, signals a characteristic outcome for the protein; Ub chain architecture is the subject of this project. The best characterized Ub types are Lys48 (K48) and Lys63 (K63) linkages; K48- and K11-linked chains target proteins for degradation by the proteasome while K63-linked chains regulate receptor endocytosis, DNA repair, oxidative stress and other pathways. Specific functions for other linkages are currently under study. Two impediments to understanding specific poly-ubiquitylated protein structures and functions are: 1) while most proteins at some point are ubiquitylated, at any given time the ubiquitylated fraction may be a small percentage of total cell protein; and 2) within the pool of ubiquitylated protein, any particular linkage may be rare. Further, very few tools are available for specific detection or purification of ubiquitylated proteins with specific poly-Ub linkages. In phase I, Ub Binding Domains (UBDs) selective for atypical poly-Ub (K6/K11/K27/K29/K33) were identified using a 150 UBD protein microarray chip developed by Dr Mark Bedford, M D Anderson Cancer Center. Also identified was the UBD with the highest selectivity for K48 linked poly-ubiquitylated proteins. In addition, KD values and specificity for novel UBDs were determined using SPR, fulfilling the aims. In Phase II LifeSensors will engineer the UBD domains to improve affinity and selectivity using UBD domains called TUBEs (Tandem Ubiquitin Binding Entities); these include nonselective as well as Linear [M1], K48- and K63-specific TUBEs. Dr. Bedford will continue his collaboration in Phase II. Also participating in Phase II will be Dr. David Fushman, University of Maryland, who has pioneered solution NMR methods to study poly-Ub chains. the Phase II project entails establishing the poly-Ub chain linkage selectivity of UBDs identified in Phase I using structure-based protein engineering and generating poly-Ub chain- selective TUBEs to improve affinity, enhance avidity of binding, and improve linkage selectivity. Finally, these novel atypical poly-Ub TUBEs will be validated for application in neuronal cells by simulating proteinopathies. .

Public Health Relevance Statement:
Tools are needed to establish the etiology of Alzheimer’s disease (AD), whose signatures appear before the disease presents. Changes in ubiquitylation patterns are among the first signatures observed, and LifeSensors is developing a method to detect these biomarkers of AD in neuronal cells; this method may be used in early detection and diagnosis of AD.

Project Terms:
Affinity; Alzheimer's Disease; Alzheimer’s disease biomarker; amino group; Amyloid beta-Protein; Architecture; Autophagocytosis; Autophagosome; Avidity; base; Binding; biological adaptation to stress; Biological Markers; C-terminal; Cancer Center; carboxylate; Cells; Cellular biology; Characteristics; Collaborations; Communities; Detection; Development; Diagnosis; Disease; DNA Repair; Doctor of Medicine; drug discovery; Early Diagnosis; Endocytosis; Engineering; Etiology; Excision; Exhibits; Functional disorder; Glycine; Goals; Grant; Imaging Device; Immune response; improved; Link; Lysine; Lysosomes; Maryland; Mediating; Methods; misfolded protein; Molecular Conformation; Molecular Profiling; multicatalytic endopeptidase complex; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; novel; Outcome; Oxidative Stress; Pathway interactions; Pattern; Phase; Plant Roots; Play; Polymers; Polyubiquitin; professor; Progress Reports; protein aggregate; Protein Binding Domain; protein degradation; Protein Engineering; protein function; Protein Microchips; protein structure function; protein transport; Proteins; receptor; receptor recycling; Reporting; response; Role; Signal Transduction; Specificity; Structure; System; tau Proteins; Texas; Time; tool; Ubiquitin; Universities