Protein Synthesis and Quality Control
I'm a paragraph. Click here to add your own text and edit me. It’s easy.
Figure 1: Nascent Protein Folding 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 (Figure 1). 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
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 (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.
Figure 2: Ubiquitination and Deubiquitination of Tail-Anchored Membrane Proteins. (A) Co-localization of deubiquitinase USP20 with the ER marker protein PDI. (B) Purified USP20 but not mutant USP20 removes ubiquitin chain from TA proteins. (C) A model of targeting and insertion of ubiquitinated TA proteins.
Recent studies from our lab discovered that newly synthesized TA membrane proteins are efficiently recognized and polyubiquitinated by cytosolic quality control factors (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 (Figure 2C). The ER-localized deubiquitinases, USP20 and USP33, remove ubiquitin chains from TA proteins (Figure 2A, B). 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 remove 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 the largest intracellular compartment 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 molecule that detects misfolded proteins in the ER and activates the XBP1 transcription factor to increase chaperones in the ER, thus mitigating ER stress (Figure 3C). 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.
Figure 3: A stress sensing IRE1/Sec61/Sec63 complex in the ER. (A) Identification of IRE1 interaction with the Sec61 translocon complex. (B) Clustering of IRE1 with the Sec61 translocon under high ER stress conditions. (C) Under mild ER stress, IRE1 activity is controlled by Sec61/Sec63-recruited luminal chaperone BiP ATPase, whereas under high ER stress, IRE1 forms higher-order oligomers, thus inducing cell death by poorly understood mechanism.
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 the Sec61/Sec63 protein translocation channel to which its substrate XBP1u mRNA is recruited by the SRP pathway (Figure 3A; 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 (Figure 3C). 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?