A major goal of our research is to understand the biogenesis of two post Golgi storage compartments: secretory granules and melanosomes. Why is this important to know?

Beta cells of pancreatic islets synthesize and maintain a very large population of insulin storage granules. When a normal person eats a meal, pancreatic islets respond to the metabolic need by releasing insulin from stores within the secretory granules of beta cells. Type I diabetes is characterized by an immune destruction of islets. Current treatments for this disease relying on glucose monitoring and insulin injection remain suboptimal and significant morbidity and mortality is still observed --children in particular are at the greatest potential risk for ultimately succumbing to serious complications. The recent advances made in islet transplantation protocols have rekindled the hope that this may be a feasible approach to restore insulin release to normal levels. Since the supply of human organs available for transplantation is unfortunately limited, implantation of engineered ß-cells may have to serve as the alternative to islets. Recent approaches inducing stem cells, pancreatic duct cells, or epithelial progenitor cells from human fetal liver to differentiate into insulin producing cells have shown promise. Nevertheless, in spite of these remarkable advances, designing cells with the function of normal beta cells has been hindered by the lack of information of what the components of a fully functional, regulated storage compartment are. Furthermore, much of the existing work on the composition of granules done in immortalized cell lines or rodent tissues may not faithfully reflect the composition of granules in human islets. Protein mass spectrometry and the expanded availability of cDNA tools have made it feasible to describe the proteome of tissues. One major project in the laboratory is to characterize the proteome of secretory granules, the organelle relevant to the development of diabetes and to do these studies in human beta cells.

Melanocytes, a cell type uniquely designed for synthesizing radiation-absorbing melanin pigments, produce a specialized organelle for this purpose, the melanosome. Melanosomes, however, are poorly understood and even less is known about proteins forming the structural matrix. This is obviously an important question as the matrix proteins are known to be essential for the specialized function of the melanocyte: to synthesize pigment and shield the skin from the damaging effects of the sun. Transformation of melanocytes to malignant melanoma is a disease process known to be accompanied by marked alteration of the melanosomal matrix and differences in melanosomal membrane content but the details of this process are not available.

Only one lumenal melanosomal matrix protein has thus far been identified at the molecular level, although others are thought to exist. Currently, we are using an in vitro melanization assay to identify other candidate proteins. By incubating melanosome enriched fractions in the presence of the melanin precursor DOPA, melanin polymers are deposited on proteins that make up the internal structure of the melanosome, blocking the ability of these lumenal proteins to be solubilized upon detergent extraction. Preliminary studies have detected several new candidate matrix proteins and one of these bands has been analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectroscopy (MS). Next, pigmentation defects (i.e. Dominant white), specifically traceable to perturbed organization of the luminal matrix have been described and to further characterize the role of the striations in melanin storage, we are analyzing the product of the Dominant white locus, Mmp115.

Am J Pathol. 2013 Mar;182(3):886-94. doi: 10.1016/j.ajpath.2012.11.027. Epub 2013 Jan 12.

Alterations in glucose homeostasis in a murine model of Chagas disease.

Nagajyothi F, Kuliawat R, Kusminski CM, Machado FS, Desruisseaux MS, Zhao D, Schwartz GJ, Huang H, Albanese C, Lisanti MP, Singh R, Li F, Weiss LM, Factor SM, Pessin JE, Scherer PE, Tanowitz HB.

Source

Division of Parasitology and Tropical Medicine, Department of Pathology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Abstract

Chagas disease, caused by Trypanosoma cruzi, is an important cause of morbidity and mortality primarily resulting from cardiac dysfunction, although T. cruzi infection results in inflammation and cell destruction in many organs. We found that T. cruzi (Brazil strain) infection of mice results in pancreatic inflammation and parasitism within pancreatic β-cells with apparent sparing of α cells and leads to the disruption of pancreatic islet architecture, β-cell dysfunction, and surprisingly, hypoglycemia. Blood glucose and insulin levels were reduced in infected mice during acute infection and insulin levels remained low into the chronic phase. In response to the hypoglycemia, glucagon levels 30 days postinfection were elevated, indicating normal α-cell function. Administration of L-arginine and a β-adrenergic receptor agonist (CL316, 243, respectively) resulted in a diminished insulin response during the acute and chronic phases. Insulin granules were docked, but the lack of insulin secretion suggested an inability of granules to fuse at the plasma membrane of pancreatic β-cells. In the liver, there was a concomitant reduced expression of glucose-6-phosphatase mRNA and glucose production from pyruvate (pyruvate tolerance test), demonstrating defective hepatic gluconeogenesis as a cause for the T. cruzi-induced hypoglycemia, despite reduced insulin, but elevated glucagon levels. The data establishes a complex, multi-tissue relationship between T. cruzi infection, Chagas disease, and host glucose homeostasis.

A mutation within the transmembrane domain of melanosomal protein Silver (Pmel17) changes lumenal fragment interactions.

Kuliawat R, Santambrogio L.

Source

Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA. kuliawat@aecom.yu.edu

Abstract

Melanocytes synthesize and store melanin within tissue-specific organelles, the melanosomes. Melanin deposition takes place along fibrils found within these organelles and fibril formation is known to depend on trafficking of the membrane glycoprotein Silver/Pmel17. However, correctly targeted, full-length Silver/Pmel17 cannot form fibers. Proteolytic processing in endosomal compartments and the generation of a lumenal Malpha fragment that is incorporated into amyloid-like structures is also essential. Dominant White (DWhite), a mutant form of Silver/Pmel17 first described in chicken, causes disorganized fibers and severe hypopigmentation due to melanocyte death. Surprisingly, the DWhite mutation is an insertion of three amino acids into the transmembrane domain; the DWhite-Malpha fragment is unaffected. To determine the functional importance of the transmembrane domain in organized fibril assembly, we investigated membrane trafficking and multimerization of Silver/Pmel17/DWhite proteins. We demonstrate that the DWhite mutation changes lipid interactions and disulfide bond-mediated associations of lumenal domains. Thus, partitioning into membrane microdomains and effects on conformation explain how the transmembrane region may contribute to the structural integrity of Silver/Pmel17 oligomers or influence toxic, amyloidogenic properties.

Mol Biol Cell. 2004 Apr;15(4):1690-701. Epub 2004 Jan 23.

Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic beta-cells.

Kuliawat R, Kalinina E, Bock J, Fricker L, McGraw TE, Kim SR, Zhong J, Scheller R, Arvan P.

Source

Division of Endocrinology and Department of Developmental/Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Abstract

In pancreatic beta-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 beta-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of beta-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.