Xia Li (Lisa)

All Publications

• Channel width modulates the permeability of DNA origami based nuclear pore mimics. bioRxiv : the preprint server for biology Feng, Q., Saladin, M., Wu, C., Cao, E., Zheng, W., Zhang, A., Bhardwaj, P., Li, X., Shen, Q., Kapinos, L. E., Mariappan, M., Lusk, C. P., Xiong, Y., Lim, R. Y., Lin, C. 2024

  Abstract

  Nucleoporins (nups) in the central channel of nuclear pore complexes (NPCs) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptors (NTRs) with bound cargos. The complex molecular interactions between nups and NTRs have been thought to underlie the gatekeeping function of the NPC. Recent studies have shown considerable variation in NPC diameter but how altering NPC diameter might impact the selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangement to mimic NPCs of different diameters. We use hepatitis B virus (HBV) capsids as a model for large-size cargos. We find that Nup62 proteins form a dynamic cross-channel meshwork impermeable to HBV capsids when grafted on the interior of 60-nm wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importing substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through NPC mimics containing Nup62 and Nup153. Our study shows the transport channel width is critical to the permeability of nup barriers and underscores the role of NTRs in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

  View details for DOI 10.1101/2024.05.09.593438

  View details for PubMedID 38766144

  View details for PubMedCentralID PMC11100828

• Nascent Chain Ubiquitination is Uncoupled from Degradation to Enable Protein Maturation. bioRxiv : the preprint server for biology Li, X., Mariappan, M. 2023

  Abstract

  A significant proportion of nascent proteins undergo polyubiquitination on ribosomes in mammalian cells, yet the fate of these proteins remains elusive. The ribosome-associated quality control (RQC) is a mechanism that mediates the ubiquitination of nascent chains on stalled ribosomes. Here, we find that nascent proteins ubiquitinated on stalled ribosomes by the RQC E3 ligase LTN1 are insufficient for proteasomal degradation. Our biochemical reconstitution studies reveal that ubiquitinated nascent chains are promptly deubiquitinated in the cytosol upon release from stalled ribosomes, as they are no longer associated with LTN1 E3 ligase for continuous ubiquitination to compete with cytosolic deubiquitinases. These deubiquitinated nascent chains can mature into stable proteins. However, if they misfold and expose a degradation signal, the cytosolic quality control recognizes them for re-ubiquitination and subsequent proteasomal degradation. Thus, our findings suggest that cycles of ubiquitination and deubiquitination spare foldable nascent proteins while ensuring the degradation of terminally misfolded proteins.

  View details for DOI 10.1101/2023.10.09.561585

  View details for PubMedID 37873109

  View details for PubMedCentralID PMC10592752

• Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy. Cell calcium Thoduvayil, S., Weerakkody, J. S., Sundaram, R. V., Topper, M., Bera, M., Coleman, J., Li, X., Mariappan, M., Ramakrishnan, S. 2023; 113: 102766

  Abstract

  High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.

  View details for DOI 10.1016/j.ceca.2023.102766

  View details for PubMedID 37295201

  View details for PubMedCentralID PMC10450995

• Signal sequences encode information for protein folding in the endoplasmic reticulum. The Journal of cell biology Sun, S., Li, X., Mariappan, M. 2023; 222 (1)

  Abstract

  One-third of newly synthesized proteins in mammals are translocated into the endoplasmic reticulum (ER) through the Sec61 translocon. How protein translocation coordinates with chaperone availability in the ER to promote protein folding remains unclear. We find that marginally hydrophobic signal sequences and transmembrane domains cause transient retention at the Sec61 translocon and require the luminal BiP chaperone for efficient protein translocation. Using a substrate-trapping proteomic approach, we identify that nascent proteins bearing marginally hydrophobic signal sequences accumulate on the cytosolic side of the Sec61 translocon. Sec63 is co-translationally recruited to the translocation site and mediates BiP binding to incoming polypeptides. BiP binding not only releases translocationally paused nascent chains but also ensures protein folding in the ER. Increasing hydrophobicity of signal sequences bypasses Sec63/BiP-dependent translocation, but translocated proteins are prone to misfold and aggregate in the ER under limited BiP availability. Thus, the signal sequence-guided protein folding may explain why signal sequences are diverse and use multiple protein translocation pathways.

  View details for DOI 10.1083/jcb.202203070

  View details for PubMedID 36459117

  View details for PubMedCentralID PMC9723807

• A second chance for protein targeting/folding: Ubiquitination and deubiquitination of nascent proteins. BioEssays : news and reviews in molecular, cellular and developmental biology Culver, J. A., Li, X., Jordan, M., Mariappan, M. 2022; 44 (6): e2200014

  Abstract

  Molecular chaperones in cells constantly monitor and bind to exposed hydrophobicity in newly synthesized proteins and assist them in folding or targeting to cellular membranes for insertion. However, proteins can be misfolded or mistargeted, which often causes hydrophobic amino acids to be exposed to the aqueous cytosol. Again, chaperones recognize exposed hydrophobicity in these proteins to prevent nonspecific interactions and aggregation, which are harmful to cells. The chaperone-bound misfolded proteins are then decorated with ubiquitin chains denoting them for proteasomal degradation. It remains enigmatic how molecular chaperones can mediate both maturation of nascent proteins and ubiquitination of misfolded proteins solely based on their exposed hydrophobic signals. In this review, we propose a dynamic ubiquitination and deubiquitination model in which ubiquitination of newly synthesized proteins serves as a "fix me" signal for either refolding of soluble proteins or retargeting of membrane proteins with the help of chaperones and deubiquitinases. Such a model would provide additional time for aberrant nascent proteins to fold or route for membrane insertion, thus avoiding excessive protein degradation and saving cellular energy spent on protein synthesis. Also see the video abstract here: https://youtu.be/gkElfmqaKG4.

  View details for DOI 10.1002/bies.202200014

  View details for PubMedID 35357021

  View details for PubMedCentralID PMC9133216

• A Molecular Mechanism for Turning Off IRE1α Signaling during Endoplasmic Reticulum Stress. Cell reports Li, X., Sun, S., Appathurai, S., Sundaram, A., Plumb, R., Mariappan, M. 2020; 33 (13): 108563

  Abstract

  Misfolded proteins in the endoplasmic reticulum (ER) activate IRE1α endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress, thereby attenuating IRE1α. Although sustained activation of IRE1α is linked to human diseases, it is not clear how IRE1α is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1α/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1α. This leads to inhibition of higher-order oligomerization and attenuation of IRE1α RNase activity during prolonged ER stress. In Sec63-deficient cells, IRE1α remains activated for a long period of time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to regulate the dynamics of IRE1α signaling in cells.

  View details for DOI 10.1016/j.celrep.2020.108563

  View details for PubMedID 33378667

  View details for PubMedCentralID PMC7809255

• Transmembrane E3 ligase RNF183 mediates ER stress-induced apoptosis by degrading Bcl-xL. Proceedings of the National Academy of Sciences of the United States of America Wu, Y., Li, X., Jia, J., Zhang, Y., Li, J., Zhu, Z., Wang, H., Tang, J., Hu, J. 2018; 115 (12): E2762-E2771

  Abstract

  The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and triggers the unfolded protein response (UPR). Failure to resolve ER stress leads to apoptotic cell death via a yet unclear mechanism. Here, we show that RNF183, a membrane-spanning RING finger protein, localizes to the ER and exhibits classic E3 ligase activities. Sustained ER stress induced by different treatments increases RNF183 protein levels posttranscriptionally in an IRE1α-dependent manner. Activated IRE1 reduces the level of miR-7, which increases the stability of RNF183 transcripts. In addition, overexpression of RNF183 leads to increased apoptosis and its depletion alleviates ER stress-induced apoptosis. Furthermore, RNF183 interacts with Bcl-xL, an antiapoptotic member of the Bcl-2 family, and polyubiquitinates Bcl-xL for degradation. Thus, RNF183 plays an important role in executing programmed cell death upon prolonged ER stress, likely by inducing apoptosis through Bcl-xL.

  View details for DOI 10.1073/pnas.1716439115

  View details for PubMedID 29507230

  View details for PubMedCentralID PMC5866564