Research & Publications
Protein synthesis and quality control:
Proteins are versatile macromolecules and are responsible for almost all cellular functions. Every human cell contains roughly 2 billion protein molecules. Cells use up to 75% of the total cellular energy budget to maintain proteome at this scale. Mariappan lab seeks to understand how cells ensure accurate targeting and folding of newly synthesized proteins. Also, we investigate how cells detect and eliminate misfolded proteins that cause human diseases, including Parkinson’s and Alzheimer's diseases. To this end, we use a multidisciplinary approach that employs biochemical assays in both in vitro and tissue culture cells, imaging, proteomics, and structural analyses
Extensive Research Description
Protein triage: Targeting versus Degradation: Membrane proteins are essential for eukaryotic life, but there are challenges particular to the synthesis and insertion of membrane proteins Membrane proteins contain hydrophobic transmembrane domains (TMDs) that typically reside within a membrane and are thus shielded from the aqueous cytosol; however, nearly all membrane proteins begin their synthesis in the cytosol. This raises the problem of exposing hydrophobic TMDs to cytosolic quality control pathways, which typically recognize hydrophobic patches present in misfolded proteins for degradation. How does quality control spare hydrophobic membrane protein but degrade misfolded proteins that expose hydrophobic patches? Imbalances in protein triage are associated with protein misfolding diseases, including prion and Parkinson’s diseases. We investigate this problem using tail-anchored (TA) proteins, an important class of membrane proteins We investigate this problem using tail-anchored (TA) proteins, an important class of membrane proteins (Mariappan et al., Nature 2010 and Mariappan et al., Nature 2011). TA proteins have a single C-terminal hydrophobic transmembrane domain (TMD) that is post-translationally targeted and inserted into the ER, mitochondria, or peroxisomes.
Our recent studies showed that newly synthesized TA membrane proteins are efficiently recognized and polyubiquitinated by cytosolic quality control (Culver and Mariappan, Journal of Cell Biology 2021). Surprisingly, polyubiquitinated TA proteins are not targeted to the proteasome for degradation but instead, they are properly targeted to the ER membrane for insertion. The ER-localized deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins. We are currently interested in addressing the following questions.
- What is the role of ubiquitination in regulating solubility and folding of membrane proteins?
- Why are ubiquitinated TA proteins not recognized by the proteasome for degradation?
- How does the ER-localized USP20/33 and removes the ubiquitin chains from TA proteins?
- What is the identity of deubiquitinase that removes ubiquitin chains from mitochondrial membrane proteins?
ER stress and the unfolded protein response (UPR): The newly synthesized proteins must fold into three-dimensional structures in order to carry out their designated functions. However, protein folding is often disrupted by external and endogenous stress conditions. We are interested in understanding mechanisms that ensure proper protein folding under stress conditions. We investigate this problem using the endoplasmic reticulum (ER), which is responsible for synthesizing and folding nearly one-third of all human proteins including antibodies, growth hormones, and membrane receptors. The unfolded protein response (UPR) of the ER plays a major role in adjusting the protein folding capacity of the ER to incoming protein load. IRE1 is the conserved UPR sensor that detects misfolded proteins in the ER and activates the XBP1 transcription factor to increase chaperones in the ER, thus mitigating ER stress. If ER stress is not mitigated, IRE1 also can mediate cell death by less understood mechanisms. IRE1-mediated cell death is implicated in the pathology of many human diseases including type 2 diabetes and neurodegenerative diseases.
For years, it has been assumed that IRE1 functions as an independent molecule to detect misfolded proteins in the ER and elicit an ER stress response. Recent studies from our laboratory discovered that IRE1 exists in a complex with Sec61/Sec63 protein translocation channel to which its substrate XBP1u mRNA is recruited by the SRP pathway (Plumb et al., eLIFE 2015; Sundaram et al., eLIFE 2017). We have recently shown that Sec61/Sec63 recruits and activates BiP ATPase to bind onto IRE1, resulting in suppression of IRE1 oligomerization and activity during mild ER stress conditions (Li et al., Cell Reports 2020). However, in the absence of IRE1 interaction with Sec61/63 or during high ER stress, IRE1 is hyperactivated by forming higher-order oligomers or clusters, thus inducing cell death in pancreatic beta cells. Currently, we are addressing the following questions:
- How does the IRE1/Sec61/Sec63 complex monitor protein translocation into the ER?
- How does the Sec61/Sec63 complex help IRE1 to make life or death decisions during ER stress?
- What is the structural architecture of the IRE1/Sec61/Sec63 complex?
Endoplasmic Reticulum; Quality Control; Protein Folding; Neurodegenerative Diseases; Unfolded Protein Response