Chemiosmotic Properties of Isolated Secretory Granules from Parotid Glands of Normal and Isoproterenol Treated Rats

Secretory granule fractions were prepared isoosmotically from the parotid glands of normal rats and from rats that had received repeated injections of isoproterenol. These granules, purified extensively, are stable after isolation, making it possible to study biophysical parameters which may relate to their role in intracellular transport and secretion.Normal parotid granules have an internal pH of (TURN)6.8 and a low ionic permeability with respect to internal buffering capacity. By contrast, granules from the treated rats have an internal pH ranging from (TURN)7.7 in (0.3 M) sucrose media to (TURN)7.1 in (100 mM) KCl media; suggesting a large ionic permeability with respect to buffering capacity. These intragranular pH values are the highest reported for any secretory vesicle type. The major content protein within control granules is (alpha)-amylase (pI (LESSTHEQ) 7), while the major species in granules from the treated rats are the proline-rich proteins (pI \u3e 10). Intragranular pH values probably differ because of these compositional differences.Normal parotid granules have a low H(\u27+) permeability and cannot utilize ATP either to acidify their interior or to create an inside-positive membrane potential. ATP-stimulated parotid granule lysis (cited by others as evidence implicating a granule H(\u27+)-ATPase) is mimicked entirely by non-hydrolyzable ATP analogs, and thus most likely does not depend on ATP hydrolysis.Parotid granules isolated from isoproterenol-treated rats, however, exhibit a measurable ATP-dependent acidification which is abolished by proton ionophores. The granules are also able to utilize ATP in creating a more inside-positive membrane potential. These effects depend on ATP hydrolysis.The underlying mechanism which permits the expression of electrogenic H(\u27+)-translocating ATPase activity in these granules (but fails to do so in the case of normal parotid granules) has not yet been found. An hypothesis is presented from which a strategy in approaching this question may be planned

86th Annual Meeting of the American Thyroid Association

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140274/1/thy.2016.29024.ata.pd

Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes

Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY

Structural Features of Vps35p Involved in Interaction with Other Subunits of the Retromer Complex

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73789/1/j.1600-0854.2007.00659.x.pd

Structural Features of Thyroglobulin Linked to Protein Trafficking

Thyroglobulin must pass endoplasmic reticulum (ER) quality control to become secreted for thyroid hormone synthesis. Defective thyroglobulin, blocked in trafficking, can cause hypothyroidism. Thyroglobulin is a large protein (~2750 residues) spanning regions I–II–III plus a C-terminal cholinesterase-like domain. The cholinesterase-like domain functions as an intramolecular chaperone for regions I–II–III, but the folding pathway leading to successful thyroglobulin trafficking remains largely unknown. Here, informed by the recent three-dimensional structure of thyroglobulin as determined by cryo-electron microscopy, we have bioengineered three novel classes of mutants yielding three entirely distinct quality control phenotypes. Specifically, upon expressing recombinant thyroglobulin, we find that first, mutations eliminating a disulfide bond enclosing a 200-amino acid loop in region I have surprisingly little impact on the ability of thyroglobulin to fold to a secretion-competent state. Next, we have identified a mutation on the surface of the cholinesterase-like domain that has no discernible effect on regional folding yet affects contact between distinct regions and thereby triggers impairment in the trafficking of full-length thyroglobulin. Finally, we have probed a conserved disulfide in the cholinesterase-like domain that interferes dramatically with local folding, and this defect then impacts on global folding, blocking the entire thyroglobulin in the ER. These data highlight variants with distinct effects on ER quality control, inhibiting domain-specific folding; folding via regional contact; neither; or both

Estrogens promote misfolded proinsulin degradation to protect insulin production and delay diabetes

Summary: Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and β cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of β cells in females but not in males. : Estrogens prevent diabetes in women, but the mechanism is poorly understood. Xu et al. report that estrogens activate the endoplasmic-reticulum-associated protein degradation pathway, which promotes misfolded proinsulin degradation, suppresses endoplasmic reticulum stress, and protects insulin secretion in mice and in human pancreatic β cells. Keywords: estrogens, beta cell, islet, endoplasmic reticulum stress, proinsulin misfolding, diabetes, bazedoxifene, sex dimorphism, ERAD, SER