The differentiation of a B cell to a plasma cell represents one of the most dramatic changes in cellular architecture known. The massive increase in the secretory pathway that occurs is necessary to allow the plasma cell to become a factory dedicated to the synthesis, assembly and transport of immunoglobulin (Ig) molecules. The production of these heteromeric proteins in the endoplasmic reticulum (ER) is both aided and monitored by a group of resident ER proteins known as molecular chaperones. If the Ig protein fails to mature properly, it is identified and transferred back across the ER membrane to the cytosol for degradation by the 26S proteasome. In addition to the massive number of Ig molecules that are processed each minute in a plasma cell, the mechanisms for generating antibody diversity put further demands on ER quality control systems in B lineage cells. Thus it is not surprising that many components of this system were first identified in immune cells, and it is possible that unique elements of the ER quality control apparatus could exist in these cells. To better define the mechanisms governing the biosynthesis of Ig proteins, we continue our studies on the molecular chaperone BiP, which binds to free Ig heavy chains (HC) and prevents their transport until they assemble with light chains (LC). We hypothesize that BiP and its co-factors work together in a carefully orchestrated fashion to aid Ig assembly, monitor the success of this operation, and finally to target improperly folded or assembled Ig subunits for degradation. Furthermore, we hypothesize that distinct regions of the ER exist to accommodate the seemingly antagonistic functions of protein folding and degradation and that individual ERdj family members allow BiP to participate in these different functions. In the present proposal we wish to further delineate critical checkpoints in Ig assembly and determine the mechanisms by which they are executed. To do so, we will determine the function of three ER localized DnaJ homologues in Ig folding, assembly and turnover, define the specificity of nucleotide exchange factors in releasing BiP from unfolded proteins, and finally delineate mechanisms used to identify unassembled Ig molecules and target them for degradation.

Public Health Relevance

Cellular processes that aid and monitor the folding and assembly of antibodies are crucial to the development of the immune system. In addition, antibodies have been unusually good substrates for identifying components of the cellular quality control machinery and should continue to provide new insights into this complex process. Abnormalities in protein folding and ER quality control can have devastating consequences as is observed in cystic fibrosis, Alzheimer's disease and prion diseases. Thus, a better understanding of the processes being investigated in this proposal is likely to have more global implications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054068-16
Application #
8291321
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Marino, Pamela
Project Start
1996-04-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
16
Fiscal Year
2012
Total Cost
$386,944
Indirect Cost
$156,620
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Otero, Joel H; Lizák, Beata; Feige, Matthias J et al. (2014) Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum. J Biol Chem 289:27504-12
Feige, Matthias J; Gräwert, Melissa A; Marcinowski, Moritz et al. (2014) The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins. Proc Natl Acad Sci U S A 111:8155-60
Behnke, Julia; Hendershot, Linda M (2014) The large Hsp70 Grp170 binds to unfolded protein substrates in vivo with a regulation distinct from conventional Hsp70s. J Biol Chem 289:2899-907
Feige, Matthias J; Hendershot, Linda M (2013) Quality control of integral membrane proteins by assembly-dependent membrane integration. Mol Cell 51:297-309
Howes, Jennifer; Shimizu, Yuichiro; Feige, Matthias J et al. (2012) C-terminal mutations destabilize SIL1/BAP and can cause Marinesco-Sjogren syndrome. J Biol Chem 287:8552-60
Lai, Chunwei Walter; Otero, Joel H; Hendershot, Linda M et al. (2012) ERdj4 protein is a soluble endoplasmic reticulum (ER) DnaJ family protein that interacts with ER-associated degradation machinery. J Biol Chem 287:7969-78
Santiago, Teresa; Kulemzin, Sergei V; Reshetnikova, Evdokia S et al. (2011) FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA. Int Immunol 23:43-53
Feige, Matthias J; Hendershot, Linda M (2011) Disulfide bonds in ER protein folding and homeostasis. Curr Opin Cell Biol 23:167-75
Vembar, Shruthi S; Jonikas, Martin C; Hendershot, Linda M et al. (2010) J domain co-chaperone specificity defines the role of BiP during protein translocation. J Biol Chem 285:22484-94
Otero, Joel H; Lizak, Beata; Hendershot, Linda M (2010) Life and death of a BiP substrate. Semin Cell Dev Biol 21:472-8

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