The long term goal of these studies is to understand general principles that govern normal and pathological CFTR folding in the endoplasmic reticulum (ER) membrane. Molecular mechanisms of membrane protein biogenesis represent a poorly understood area of biology with major implications for human health and disease. Cystic fibrosis (CF) is one such example where inherited mutations give rise to abnormally folded conformers that are rapidly recognized and degraded by cellular quality control machinery. Evidence now indicates that the primary defect in up to 90% of the 30,000 CF patients in the US is caused by deletion of a single phenylalanine residue at position 508. This causes a subtle disruption of the early folding pathway in the ER and prevents proper association of membrane-bound and cytosolic domains. A major limitation in understanding CF and related disorders is that many aspects of folding occur coincident with synthesis in a biochemically complex environment comprised of the translating ribosome and the Sec61 ER biosynthetic machinery. Therefore, traditional biochemical and biophysical tools are poorly suited to study cotranslational folding events. Experiments in this proposal will take advantage of recent developments that now provide direct access to structural features of the nascent polypeptide in its native folding environment. Fluorescent and photoactive probes will be incorporated into uniform cohorts of programmed translocation intermediates using synthetic modified aminoacyl-tRNAs. Photocrosslinking, fluorescence quenching and fluorescence resonance energy transfer (FRET) will then be used to address three fundamental problems faced by all native membrane proteins. Using wild type and disease related CFTR mutants, we will first define how structural features within the nascent polypeptide control the translocation pathway and establish transmembrane topology and membrane integration by regulating nascent chain exposure to cytosolic and lumenal compartments. Second, we will determine when during synthesis, and where within the translocation pathway, nascent 2? structure begins to collapse and how 2? structure formation influences translocon gating dynamics. Third, we will define cotranaslational folding events that give rise to 3? structure and determine how inherited mutations disrupt this process in CF disease. This work will contribute significantly to our understanding of the molecular pathogenesis of CF and provide a general framework with which to pharmacologically manipulate physiological and pathological parameters of protein folding disorders. Relevance of the proposed research to human health: Disorders of membrane protein folding represent a rapidly expanding area of medicine that affects tens of thousands of Americans at enormous economic and social cost. Treatments for these disorders have been limited because basic understanding of biological folding pathways remain largely unknown. To overcome this problem, this project will use novel biophysical approaches to define when transmembrane segments begin to fold in the context of ER biosynthetic machinery, how they are inserted into the ER membrane, and the specific steps at which folding is disrupted by inherited disease- related mutations. .

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK051818-13
Application #
8246410
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Mckeon, Catherine T
Project Start
1996-12-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
13
Fiscal Year
2012
Total Cost
$301,871
Indirect Cost
$105,851
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
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Matsumura, Yoshihiro; Sakai, Juro; Skach, William R (2013) Endoplasmic reticulum protein quality control is determined by cooperative interactions between Hsp/c70 protein and the CHIP E3 ligase. J Biol Chem 288:31069-79
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Khushoo, Amardeep; Yang, Zhongying; Johnson, Arthur E et al. (2011) Ligand-driven vectorial folding of ribosome-bound human CFTR NBD1. Mol Cell 41:682-92
Devaraneni, Prasanna K; Conti, Brian; Matsumura, Yoshihiro et al. (2011) Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex. Cell 146:134-47
Matsumura, Yoshihiro; David, Larry L; Skach, William R (2011) Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum-associated degradation. Mol Biol Cell 22:2797-809
Brodsky, Jeffrey L; Skach, William R (2011) Protein folding and quality control in the endoplasmic reticulum: Recent lessons from yeast and mammalian cell systems. Curr Opin Cell Biol 23:464-75

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