Autophagy is responsible for the vesicular sequestration and lysosomal degradation of unwanted, damaged or harmful macromolecular cytoplasmic components, resulting in nutrient recycling. Thus, autophagy plays an important role in a wide range of organismal homeostasis processes including life-span extension, embryonic development, tissue-differentiation, cell-growth, surviving environmental stress conditions like starvation, high temperatures and hypoxia as well as internal stressors like damaged organelles, mutant proteins, free radicals and intracellular pathogens. Defects in autophagy are implicated in many disorders such as reduced life-span, neurodegenerative and muscular disorders, age-weakened immunity against infectious diseases;hence, elucidating the structure-based mechanism of autophagy proteins is essential to understanding these diseases and devising potential therapeutics targeting them. Intrinsically disordered regions (IDRs), i.e. regions that lack ordered secondary and tertiary structure, are common, yet poorly understood, features of many proteins. Mutations implicated in important diseases like neurodegenerative disorders like Alzheimer's disease often map to IDRs. Our bioinformatics analysis shows that IDRs are a predominant feature of autophagy proteins. We experimentally verify structural disorder in a selected subset of autophagy protein IDRs, including a large IDR within the conserved autophagy effector, Beclin 1, an essential component of the autophagy nucleation complex. Further, based on the results of a bioinformatics analyses to investigate the potential biological function of IDRs in mediating protein interactions, we experimentally investigate selected binding motifs predicted within selected IDRs, as well as conformational transitions in these IDRs, with a special focus on the Beclin 1 IDR. Further, we have identified several prospective protein interactions of the Beclin 1 IDR, and these will be experimentally validated. Beclin 1 is directly implicated in many human diseases. Reduced Beclin 1 levels are associated with decreased longevity and an elevated risk of age-related disorders like Alzheimer's disease;while an increase in Beclin 1-mediated autophagy, such as by caloric restriction, increases cellular and organismal life span. Beclin 1 appears to be a protein interaction hub for autophagy as it is implicated in interactions with at least 20 other cellular proteins. The work in this propsal will lay the groundwork for future research building a detailed structural and mechanistic understanding of these interactions that will elucidate how these interactions compete with or complement each other to regulate Beclin 1-mediated autophagy, the role of disruptions of these interactions in human diseases and potential therapeutics that target the Beclin 1 IDR or its interactions.

Public Health Relevance

Autophagy, a conserved homeostatic catabolic cellular pathway responsible for lysosomal degradation of damaged, defunct or dangerous macromolecular assemblies plays an important role in life span determination, developmental and differentiation processes;hence autophagy dysfunction is implicated in a wide range of human disorders including aging and age-related neurodegenerative and muscular disorders. Mutations implicated in important diseases like age-related dysfunctions, especially neurodegenerative disorders often map to intrinsically disordered regions, i.e. regions that lack ordered secondary and tertiary structure;and such regions appear to be a predominant feature of autophagy proteins. The long-term goal of this research is to elucidate the function of intrinsically disordered regions in autophagy, in order to devise therapeutics that can modulate levels of autophagy within cells to treat human disorders and prolong life.

Agency
National Institute of Health (NIH)
Type
Small Research Grants (R03)
Project #
1R03NS090939-01A1
Application #
8773287
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Sutherland, Margaret L
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
North Dakota State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Fargo
State
ND
Country
United States
Zip Code
58108
Glover, Karen; Mei, Yang; Sinha, Sangita C (2016) Identifying intrinsically disordered protein regions likely to undergo binding-induced helical transitions. Biochim Biophys Acta 1864:1455-63
Mei, Yang; Glover, Karen; Su, Minfei et al. (2016) Conformational flexibility of BECN1: Essential to its key role in autophagy and beyond. Protein Sci 25:1767-85
(2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1-222
Mei, Yang; Su, Minfei; Sanishvili, Ruslan et al. (2016) Identification of BECN1 and ATG14 Coiled-Coil Interface Residues That Are Important for Starvation-Induced Autophagy. Biochemistry 55:4239-53
Mei, Yang; Ramanathan, Arvind; Glover, Karen et al. (2016) Conformational Flexibility Enables the Function of a BECN1 Region Essential for Starvation-Mediated Autophagy. Biochemistry 55:1945-58
Wei, Yongjie; An, Zhenyi; Zou, Zhongju et al. (2015) The stress-responsive kinases MAPKAPK2/MAPKAPK3 activate starvation-induced autophagy through Beclin 1 phosphorylation. Elife 4:
Jensen, Jaime L; Balbo, Andrea; Neau, David B et al. (2015) Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR. Biochemistry 54:5867-77
Su, Minfei; Mei, Yang; Sanishvili, Ruslan et al. (2014) Targeting γ-herpesvirus 68 Bcl-2-mediated down-regulation of autophagy. J Biol Chem 289:8029-40
Mei, Yang; Su, Minfei; Soni, Gaurav et al. (2014) Intrinsically disordered regions in autophagy proteins. Proteins 82:565-78