Cell Biology; Endoplasmic Reticulum; Quality Control; Unfolded Protein Response; Protein Aggregates
Our laboratory is interested in understanding cellular mechanisms that prevent accumulation and aggregation of misfolded proteins in the cell. A better understanding of these mechanisms is essential for developing effective treatments for numerous human disorders including diabetes, neurodegeneration, and aging. We are specifically interested in the endoplasmic reticulum (ER) associated pathways that play a central role in preventing accumulation of misfolded proteins. The ER is the major site for synthesis, folding and maturation of secretory and membrane proteins, which account for nearly one third of the human proteome. Folding of these proteins is aided by chaperones and enzymes in the lumen of the ER. Despite this support, a significant proportion of newly synthesized proteins are misfolded in the ER due to genetic mutations, complexity of folding, and changes in the flux of proteins. Thus, the ER has evolved with two important pathways to deal with these misfolded proteins:
1) The unfolded protein response (UPR) pathway senses the misfolded proteins in the ER and induces the genes responsible for increasing ER folding capacity.
2) The ER associated degradation (ERAD) pathway routes the misfolded proteins from the ER to the cytosol for degradation by the proteasome.
We use a variety of techniques, including biochemical reconstitution, mammalian cell culture, molecular biology and cell imaging to understand molecular mechanisms involved in these pathways.
Specialized Terms: Unfolded Protein Response (UPR); Endoplasmic Reticulum; Protein Translocation and Insertion; ER stress response; Misfolded Proteins; Protein Quality Control
Extensive Research Description
1) The unfolded protein response (UPR) pathway: The accumulation of misfolded proteins in the ER (termed as “ER stress”) triggers three branches of UPR signaling (IRE1, PERK and ATF6) to restore ER homeostasis. We specifically focus on the IRE1 branch. IRE1 is a transmembrane endoribonuclease and is the most evolutionarily conserved branch of the UPR, present from yeast to humans. Upon ER stress, Ire1 becomes activated and mediates two important signaling outputs to restore ER folding homeostasis: (1) Ire1 mediates an unconventional splicing of unspliced XBP1-mRNA. This results in synthesis of an active transcription factor, XBP1s, which upregulates genes necessary for increasing the ER folding capacity. (2) Ire1 promiscuously cleaves mRNAs encoding secretory and membrane proteins through the regulated Ire1-dependent decay (RIDD) pathway, which reduces the load of incoming proteins. Under severe ER stress conditions, however, Ire1 initiates cell death by apoptosis. This phenomenon is often implicated in the loss of pancreatic β cells and neuronal cells, thus leading to the pathogenesis of diabetes and neurodegeneration, respectively. Despite these physiological and pathological roles, fundamental mechanisms and regulatory components involved in the Ire1-UPR are poorly understood. The goal of the study is to (1) identify cellular pathways that regulate the Ire1 signaling using a complementary affinity purification/mass spectrometry and genome wide siRNA screens. (2) We aim to derive mechanistic understanding of Ire1 signaling by biochemical reconstitution combined with structural studies. Our studies will advance knowledge of UPR function under basal and disease conditions, and provide insights into how UPR pathways can be targeted in therapeutic strategies.
2) The ER associated degradation (ERAD) pathway: Cell function and viability critically depend on recognition and elimination of misfolded proteins to avoid accumulation of toxic aggregates. Nowhere is the misfolding of proteins more frequent than in the ER where proteins with complex topology and multiple transmembrane domains are synthesized. These misfolded membrane proteins are destroyed via the ER associated degradation (ERAD) pathway. ERAD is associated with many human diseases, however, little is known about the molecules and mechanisms involved in the degradation of membrane proteins. For example, when and how are misfolded membrane proteins detected? How are transmembrane domains of membrane proteins extracted from membranes? How are hydrophobic transmembrane domains shielded from aggregation during targeting to the proteasome for degradation? To answer these questions and understand this process in detail, we are developing in vitro cell-free and in vivo imaging based assays with the mutant form of CFTR membrane protein that causes cystic fibrosis.
- Plumb, P., Zhang, Z. R., Appathurai, S., and Mariappan. M#. (2015) A functional link between the co-translational protein translocation pathway and the UPR. eLIFE # Corresponding author
- Mariappan, M., *Mateja, A., Dobosz, M., Bove, E., Hegde, R. S., and Keenan, R. J. (2011) The mechanisms of membrane associated steps in tail-anchored protein insertion. Nature 477, 61-66.
- Hessa, T., Sharma, A., Mariappan, M., Eshleman, H. D., Gutierrez, E., and Hegde, R. S. (2011) Protein targeting and degradation pathways are coupled for elimination of mislocalized proteins. Nature 475, 393-397.
- Mariappan, M., Li, X., Stefanovic, S., Sharma, A., Mateja, A., Keenan, R. J., and Hegde, R. S. (2010) A ribosome-associating factor chaperones tail-anchored membrane proteins. Nature 466, 1120-1124.
- Mateja, A., Szlachcic, A., Downing, M. E., Dobosz, M., Mariappan, M., Hegde, R. S., and Keenan, R. J. (2009) The structural basis of tail-anchored membrane protein recognition by Get3. Nature 461, 361-366.
- Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R., and Rudolph, M. G. (2005) Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine-generating enzyme. Cell 121, 541-552. 541-52