The overall goal of this proposal is to develop and implement a systematic approach that will result in the identification and characterization of lysosomal proteins and to understand their role in human disease. This approach relies on the finding that most lysosomal proteins contain a common post-translational modification, the mannose 6-phosphate (M6P) recognition marker, which distinguishes them from other types of proteins. There are three specific aims.
Specific Aim 1 is to identify the spectrum of M6P glycoproteins encoded by the human genome using both two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry based approaches. This will identify a number of previously undiscovered candidate lysosomal proteins and will create a proteomics resource to investigate their role in human disease.
Specific Aim 2 is to begin to characterize the function of previously unidentified M6P glycoproteins, This will entail determining their subcellular distribution to verify their lysosomal location as well as generating reagents for structural and functional studies, Specific Aim 3 is to investigate the molecular bases for lysosomal storage diseases of unknown etiology. Two approaches will be used: 1) The """"""""disease-to-protein"""""""" approach will entail global comparisons of M6P glycoproteins in normal and disease specimens using the resources created in Specific Aim 1. Extensions of this approach may be applicable towards development of clinical screening methods for lysosomal storage diseases. 2) The protein-to-disease approach will create a database of the properties of newly characterized M6P glycoproteins and use this information to identify candidates for associated diseases. Potential disease genes identified using either approach will be scrutinized using molecular genetic analysis. Given that this approach has resulted in the identification of several previously uncharacterized lysosornal proteins and the molecular basis for three hereditary diseases, it is anticipated that extensions of this discovery-based research will result in additional important findings.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054317-08
Application #
7032993
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Mckeon, Catherine T
Project Start
1999-06-15
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2008-03-31
Support Year
8
Fiscal Year
2006
Total Cost
$379,609
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Pharmacology
Type
Schools of Medicine
DUNS #
617022384
City
Piscataway
State
NJ
Country
United States
Zip Code
08854
Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline et al. (2017) Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome. Mol Cell Proteomics 16:194-212
Huang, Ling; Pike, Douglas; Sleat, David E et al. (2014) Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome. PLoS One 9:e88893
Sleat, David E; Sun, Pengling; Wiseman, Jennifer A et al. (2013) Extending the mannose 6-phosphate glycoproteome by high resolution/accuracy mass spectrometry analysis of control and acid phosphatase 5-deficient mice. Mol Cell Proteomics 12:1806-17
Sleat, David E; Wiseman, Jennifer A; Sohar, Istvan et al. (2012) Proteomic analysis of mouse models of Niemann-Pick C disease reveals alterations in the steady-state levels of lysosomal proteins within the brain. Proteomics 12:3499-509
Della Valle, Maria Cecilia; Sleat, David E; Zheng, Haiyan et al. (2011) Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes. Mol Cell Proteomics 10:M110.006403
Qian, Yi; Lee, Intaek; Lee, Wang-Sik et al. (2010) Functions of the alpha, beta, and gamma subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. J Biol Chem 285:3360-70
Lendrihas, Thomas; Hunter, Gregory A; Ferreira, Gloria C (2010) Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase. J Biol Chem 285:3351-9
Sleat, David E; Ding, Lin; Wang, Shudan et al. (2009) Mass spectrometry-based protein profiling to determine the cause of lysosomal storage diseases of unknown etiology. Mol Cell Proteomics 8:1708-18
Deng, Ling; Moore, Dirk F (2009) Composite likelihood modeling of neighboring site correlations of DNA sequence substitution rates. Stat Appl Genet Mol Biol 8:Article 6
L├╝bke, Torben; Lobel, Peter; Sleat, David E (2009) Proteomics of the lysosome. Biochim Biophys Acta 1793:625-35

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