Randal J. Kaufman

Control of mRNA translation preserves endoplasmic reticulum function in beta cells and maintains glucose homeostasis

Type 2 diabetes is a disorder of hyperglycemia resulting from failure of beta cells to produce adequate insulin to accommodate an increased metabolic demand. Here we show that regulation of mRNA translation through phosphorylation of eukaryotic initiation factor 2 (eIF2α) is essential to preserve the integrity of the endoplasmic reticulum (ER) and to increase insulin production to meet the demand imposed by a high-fat diet. Accumulation of unfolded proteins in the ER activates phosphorylation of eIF2α at Ser51 and inhibits translation. To elucidate the role of this pathway in beta-cell function we studied glucose homeostasis in Eif2s1 tm1Rjk mutant mice, which have an alanine substitution at Ser51. Heterozygous (Eif2s1 +/tm1Rjk) mice became obese and diabetic on a high-fat diet. Profound glucose intolerance resulted from reduced insulin secretion accompanied by abnormal distension of the ER lumen, defective trafficking of proinsulin, and a reduced number of insulin granules in beta cells. We propose that translational control couples insulin synthesis with folding capacity to maintain ER integrity and that this signal is essential to prevent diet-induced type 2 diabetes.

Cytoprotection by pre‐emptive conditional phosphorylation of translation initiation factor 2

Transient phosphorylation of the α‐subunit of translation initiation factor 2 (eIF2α) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2α phosphorylation‐dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2α phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress‐inducible eIF2α kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2α kinase, Fv2E‐PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E‐PERK activation enhanced the expression of numerous stress‐induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2α phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress‐induced signals and that activation of this pathway can single‐handedly promote a stress‐resistant preconditioned state.

Mannose-dependent endoplasmic reticulum (ER)-Golgi intermediate compartment-53-mediated ER to Golgi trafficking of coagulation factors V and VIII.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is the site of segregation of secretory proteins for anterograde transport, via packaging into COPII-coated transport vesicles. ERGIC-53 is a homo-hexameric transmembrane lectin localized to the ERGIC that exhibits mannose-selective properties in vitro. Null mutations in ERGIC-53 were recently shown to be responsible for the autosomal recessive bleeding disorder, combined deficiency of coagulation factors V and VIII. We have studied the effect of defective ER to Golgi cycling by ERGIC-53 on the secretion of factors V and VIII. The secretion efficiency of factor V and factor VIII was studied in a tetracycline-inducible HeLa cell line overexpressing a wild-type ERGIC-53 or a cytosolic tail mutant of ERGIC-53 (KKAA) that is unable to exit the ER due to mutation of two COOH-terminal phenylalanine residues to alanines. The results show that efficient trafficking of factors V and VIII requires a functional ERGIC-53 cycling pathway and that this trafficking is dependent on post-translational modification of a specific cluster of asparagine (N)-linked oligosaccharides to a fully glucose-trimmed, mannose9 structure.

Neuroserpin Polymers Activate NF-κB by a Calcium Signaling Pathway That Is Independent of the Unfolded Protein Response

The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies is characterized by the accumulation of ordered polymers of mutant neuroserpin within the endoplasmic reticulum of neurones. We show here that intracellular neuroserpin polymers activate NF-κB by a pathway that is independent of the IRE1, ATF6, and PERK limbs of the canonical unfolded protein response but is dependent on intracellular calcium. This pathway provides a mechanism for cells to sense and react to the accumulation of folded structures of mutant serpins within the endoplasmic reticulum. Our results provide strong support for the endoplasmic reticulum overload response being independent of the unfolded protein response.

Is the ER Stressed out in Diabetic Kidney Disease

Despite decades of concerted mechanistic research, the pathogenesis of progressive diabetic nephropathy remains uncertain. Although improved glycemic control and treatment with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers ameliorate the progression of diabetic

Protein Retention in the Endoplasmic Reticulum Mediated by GRP78

Proteins destined for secretion in mammalian cells transit the secretory pathway consisting of two major compartments: the endoplasmic reticulum (ER) and Golgi complex (GC) (Fig. 8.1). Proteins are first cotranslationally translocated into the lumen of the ER. The ER is the site of initial processing events that are crucial for proper folding of the nascent polypeptide. These processing events include signal peptide cleavage (1), addition of core N-linked oligosaccharides at consensus recognition sites (2), and disulfide bond formation mediated by protein disulfide isomerase (3). Attaining an appropriate conformation is essential for a protein to be efficiently transported from the ER to the GC (4). For most proteins, transport from the ER to the GC is the rate-limiting step for secretion (5, 6). This transport step requires ATP (7). Retention in the ER, and eventual degradation, is the fate of many secretion-incompetent proteins (8).