Investigating Mammalian Unfolded Protein Response: The Physiology And Regulatory Mechanisms Of Ire1Alpha-Xbp1 Signaling
The endoplasmic reticulum (ER) serves as the site of protein synthesis, folding, maturation, modification, secretion, and degradation for approximately one-third of the proteome. Disruptions in ER homeostasis activate an ER-tonucleus signaling pathway termed the unfolded protein response (UPR). The IRE1[alpha]-XBP1 pathway is the most conserved arm of UPR and upon activation, acts to restore ER homeostasis. Significantly, IRE1[alpha]-XBP1 dysfunction has been implicated in the development and pathogenesis of protein-misfolding diseases. Although the general events underlying mammalian IRE1[alpha]-XBP1 activation and signaling have been reported, the mechanistic details remain unclear. In addition to serving as a critical arm of UPR signaling, the IRE1[alpha]-XBP1 pathway was essential and indispensable for differentiation of pre-adipocytes into mature fat cells. Upon adipogenesis, C/EBP[beta], a key initiator of the adipogenic program, was shown to bind to the proximal promoter of the Xbp1 gene and induce its expression. XBP1, an essential UPR transcription factor, was required for the subsequent modulation of C/EBP[alpha], an adipogenic protein critical for maintaining the differentiated state. Interestingly, adipogenic differentiation was associated with a low degree of physiological UPR required for IRE1[alpha] activation and Xbp1 splicing. These novel findings positioned the IRE1[alpha]-XBP1 pathway as a critical modulator of the transcriptional cascade underlying adipocyte differentiation, thus supporting the notion of UPR in metabolic dysfunction. To further understand the activation mechanism of IRE1[alpha], a proteomicsbased mass spectrometry screen was performed to uncover novel IRE1[alpha]interacting factors that may play a role in regulating its activation and signaling. Non-muscle myosin IIB (NMIIB), a component of the cytoskeleton machinery, was identified and shown to interact specifically with IRE1[alpha] in an ER stressdependent manner. NMIIB was further characterized to be required for IRE1[alpha] activation and downstream signaling. Specifically, the motor activity of NMIIB and the actin cytoskeleton were essential in modulating IRE1[alpha] higher-order oligomer formation, a key activating step. Physiologically, NMIIB function was conserved as both mammalian cells and C. elegans lacking NMII exhibited hypersensitivity to ER stress. Collectively, the studies presented in this dissertation have contributed original and novel insight into the physiology and mechanisms underlying mammalian IRE1[alpha]-XBP1 activation and signaling.
ER stress; UPR; IRE1alpha; XBP1
Lis, John T; Brenna, James Thomas
Ph.D. of Genetics
Doctor of Philosophy
dissertation or thesis