Ultrastructural analyses also revealed an increase in the number of glucagon granules in cells of ERdj4GT/GTmice (Figure 3E)

Ultrastructural analyses also revealed an increase in the number of glucagon granules in cells of ERdj4GT/GTmice (Figure 3E). of ERdj4GT/GTmice died perinatally associated with fetal growth restriction, reduced hepatic glycogen stores, and hypoglycemia. Surviving adult mice exhibited evidence of constitutive ER stress in multiple cells/tissues, TBPB including fibroblasts, lung, kidney, salivary gland, and pancreas. Elevated ER stress in pancreatic cells of ERdj4GT/GTmice was associated with cell loss, hypoinsulinemia, and glucose intolerance. Collectively these results suggest an important role for ERdj4 in maintaining ER homeostasis during normal fetal growth and postnatal adaptation to metabolic stress. == INTRODUCTION == Nascent proteins destined for the cell surface, intracellular organelles, or secretion enter the endoplasmic reticulum (ER), where they undergo chaperone-mediated protein folding to achieve a stable conformation. Highly metabolic cells, including pancreatic acinar and cells, plasma B cells, and serous and mucous cells of the salivary gland, invoke the unfolded protein response (UPR) to increase ER folding capacity and maintain homeostasis in the face of increased protein load and consequent ER stress (Zhanget al., 2002;Iwakoshiet al., 2003;Leeet al., 2005;Iwawakiet al., 2010). The UPR reduces ER burden by attenuating protein translation and up-regulating the machinery involved in protein folding, quality control, and ER-associated degradation (ERAD). The ER transmembrane sensors protein kinase RNAlike ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) detect accumulation of unfolded or misfolded protein in the ER lumen and transduce signals across the ER membrane to alleviate ER stress. PERK activation results in phosphorylation of eukaryotic translation initiation factor 2, which inhibits translation initiation, thereby reducing the load of newly synthesized proteins within the ER. Activation of ATF6 results in relocation to the Golgi, where proteolytic cleavage releases a cytosolic ATF6 fragment that translocates to the nucleus to stimulate transcription of UPR target genes, including ER chaperones. The activated cytosolic endoribonuclease domain of IRE1 removes an unspliced intron from X-box binding protein 1 Goserelin Acetate (XBP1) mRNA, leading to translation of a transcription factor that up-regulates expression of chaperones involved in protein folding, as well as components of ERAD. Failure of the UPR to resolve ER stress can trigger apoptosis (Schroder and Kaufman, 2005;Ron and Walter, 2007). Molecular chaperones are critical components of the ER quality control machinery and distinguish TBPB among unfolded, correctly folded, and terminally misfolded proteins within the ER. Immunoglobulin-binding protein (BiP)/glucose-regulated protein 78, an ER-localized member of the heat shock protein (HSP) 70 family, is a multifunctional chaperone that assists in the folding TBPB of nascent proteins (Leeet al., 1999;Wanget al., 2005), facilitates targeting of misfolded proteins for proteasomal degradation (Nishikawaet al., 2001;Molinariet al., 2002), and plays a key role in activation of UPR sensors in response to ER stress (Bertolottiet al., 2000;Shenet al., 2002a). BiP contains a C-terminal substrate-binding domain (SBD) that binds unfolded substrates with low affinity via interaction with exposed hydrophobic regions. Hydrolysis of ATP bound to the N-terminal nucleotide-binding domain (NBD) of BiP causes high-affinity binding of substrate. Exchange of ADP for ATP releases the substrate from BiP, allowing the substrate to continue folding (Mayer and Bukau, 2005). An arginine residue located on the surface of the NBD in the ATP-bound form is critical for communication with the SBD and interaction with HSP40 cochaperones (Jianget al., 2005;Awadet al., 2008). The ER-localized DnaJ (ERdj) family of HSP40 TBPB cochaperones induce high-affinity substrate binding by interacting with the NBD of BiP and stimulating ATP hydrolysis. Whereas some ERdjs interact with BiP independently of substrate, others bind substrates directly and recruit BiP (Fewellet al., 2001;Shen and Hendershot, 2005;Jinet al., 2008;Petrovaet al., 2008). ERdjs facilitate protein folding and/or degradation of newly translocated TBPB (ERdj13), unfolded (ERdj3 and ERdj6), or misfolded (ERdj4 and ERdj5) substrates, often by interacting with translocation or ERAD machinery (Oteroet al., 2010). ERdj4 is a soluble DnaJ protein that binds BiP through a His-Pro-Asp (HPD) motif in the J domain and likely interacts with unfolded/misfolded substrates through a C-terminal glycine/phenylalanine-rich region (Shenet al., 2002b;Donget al., 2008;Laiet al., 2012). ERdj4 is ubiquitously expressed at very low levels (Weitzmannet al., 2007;Hageman and Kampinga, 2009) and is up-regulated in response ER stress (Shenet al., 2002b;Donget al., 2008) by the UPR signal transducer XBP1 (Leeet al., 2003;Kanemotoet al., 2005). ERdj4 binds to unfolded (insulin2) or misfolded (insulin2C96Y, surfactant protein Cexon4[SP-Cexon4], and SP-CL188Q) substrates to facilitate their removal from the ER for degradation by the proteasome (Donget.