Mariappan Lab Research Projects
Proteins carry out most cellular functions and are responsible for the structure, function, and regulation of all organisms on Earth. Yet, we do not fully understand how proteins are properly produced and degraded (trashed) when they become defective in mammalian cells. Failure in any of these processes can lead to devastating human diseases, including type 2 diabetes, cystic fibrosis, Alzheimer's, and Parkinson's disease. Mariappan's lab has three projects that aim to understand the protein life cycle using biochemical, fluorescence imaging, CRISPR/Cas9 editing, proteomics, and structural approaches.
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Project 1: Membrane Protein Biogenesis and Quality Control
About 30% of the human proteome consists of membrane proteins, which are crucial for cell physiology, including cell-cell communication, signal transduction, and molecular transport across membranes. Due to their high hydrophobicity, membrane proteins face challenges during biogenesis and degradation, which are further exacerbated for tail-anchored (TA) proteins, a vital class of membrane proteins. TA proteins contain a single C-terminal transmembrane domain (TMD) (Figure 1). They are post-translationally targeted and inserted into the membranes of the endoplasmic reticulum (ER), mitochondria, or peroxisomes. Our research, along with others, has identified numerous factors mediating the targeting and insertion of TA proteins into the ER membrane through the GET (Guided Entry of Tail-anchored proteins) pathway (Mariappan et al., Nature 2010; Mariappan et al., Nature 2011; Heo et al., Cell Reports 2023)
We recently discovered that newly synthesized TA proteins are polyubiquitinated in cells. Polyubiquitinated proteins, particularly those decorated with K48-linked ubiquitin chains, are typically routed to proteasomal degradation. Instead, polyubiquitinated TA proteins are properly captured by the targeting chaperone GET3 (TRC40) and delivered to the ER membrane, where the USP20/33 deubiquitinases subsequently remove their ubiquitin modifications (Culver and Mariappan Journal of Cell Biology, 2021). Our ongoing studies aim to understand why TA proteins are initially decorated with ubiquitin chains and why these modifications are later removed. What is the role of ubiquitin modification in TA protein insertion and degradation? Addressing these questions will likely explain why mutations in GET3 are associated with Parkinson's disease in humans.
Project 2: ER Stress and the Unfolded Protein Response (UPR)
Secretory and membrane proteins represent ~30% of all human proteins and are synthesized in the endoplasmic reticulum (ER). These proteins play broad roles in all aspects of intra- and extra-cellular communications, and ~70% of pharmaceutical drugs target membrane and secretory proteins. However, protein synthesis in the ER often surpasses its folding capacity, resulting in the accumulation of misfolded proteins and causing ER stress. The unfolded protein response (UPR) plays a significant role in adjusting the protein folding capacity of the ER to the incoming protein load. IRE1 is a conserved UPR sensor that detects misfolded proteins in the ER lumen and activates the transcription factor XBP1 to alleviate ER stress (Figure 2). If ER stress is not mitigated, IRE1 can also mediate cell death by less understood mechanisms, which is implicated in type 2 diabetes and neurodegenerative diseases. Therefore, it is essential to understand how IRE1 activity is regulated in human cells to develop better strategies to treat these diseases.
Studies from my laboratory discovered that IRE1 exists in a direct complex with the Sec61 protein translocation channel, to which the SRP pathway recruits its substrate XBP1 mRNA (Plumb et al., eLIFE 2015). We have further shown that the Sec61 translocon-associated protein Sec63 recruits the luminal chaperone BiP to bind onto IRE1, thus turning off IRE1 signaling once ER stress is alleviated (Sundaram et al., eLIFE 2017; Li et al., Cell Reports 2020). Without the Sec complex, IRE1 is hyperactivated and induces cell death in insulin-secreting pancreatic beta cells, which is implicated in type 2 diabetes. Our current studies focus on understanding how IRE1 makes life-or-death decisions during ER stress in neuronal and pancreatic beta cells (Figure 2). Additionally, we aim to obtain the structural information of the IRE1/Sec61/Sec63 complex to understand how IRE1 senses the accumulation of unfolded proteins in the ER.
Project 3: Protein Biogenesis and Quality Control in the Endoplasmic Reticulum:
The endoplasmic reticulum (ER) synthesizes approximately one-third of the human proteome, including secreted proteins and membrane proteins. A central challenge for the ER is matching fluctuating demands for protein synthesis with its available protein-folding capacity. Our laboratory discovered that signal sequences (SS), traditionally viewed as simple ER-targeting signals, also encode information that recruits specific chaperones required for protein folding (Sun et al., Journal of Cell Biology, 2023). We found that weak (less hydrophobic) signal sequences induce a transient pause during translocation through the Sec61 channel, allowing Sec63 to recruit the luminal Hsp70 chaperone BiP (Figure 3). This mechanism promotes efficient translocation, proper protein folding, and prevents aggregation within the ER.
Building on these findings, we recently discovered that many proteins containing weak signal sequences become trapped within the Sec61 translocon, particularly during ER stress. These stalled substrates are eliminated through a previously unrecognized quality-control pathway that preserves translocon function and ER homeostasis. Our current studies aim to identify the molecular machinery underlying this pathway and determine its relevance to protein-misfolding disorders, including Alzheimer's and prion diseases.