A low-affinity Ca2+-dependent association of calmodulin with the Rab3A effector domain inversely correlates with insulin exocytosis.

The stimulus-response coupling pathway for glucose-regulated insulin secretion has implicated a rise in cytosolic [Ca2+]i as a key factor to induce insulin exocytosis. However, it is unclear how elevated [Ca2+]i communicates with the pancreatic beta-cell's exocytotic apparatus. As Rab3A is a model protein involved in regulated exocytosis, we have focused on its role in regulating insulin exocytosis. By using a photoactivatable cross-linking synthetic peptide that mimics the effector domain of Rab3A and microsequence analysis, we found calmodulin to be a major Rab3A target effector protein in pancreatic beta-cells. Coimmunoprecipitation analysis from pancreatic islets confirmed a Rab3A-calmodulin interaction in vivo, and that it inversely correlated with insulin exocytosis. Calmodulin affected neither GTPase nor guanine nucleotide exchange activity of Rab3A. The calmodulin-Rab3A interaction was pH- and Ca2+-dependent, and it was preferential for GTP-bound Rab3A. However, Rab3A affinity for calmodulin was relatively low (Kd = 18-22 micromol/l at 10(-5) mol/l [Ca2+]) and competed by other calmodulin-binding proteins that had higher affinity (e.g., Ca2+/calmodulin-dependent protein kinase-2 [CaMK-2] [Kd = 300-400 nmol/l at 10(-5) mol/l [Ca2+]]). Moreover, the Ca2+ dependence of the calmodulin-Rab3A interaction (K0.5 = 15-18 micromol/l [Ca2+], maximal at 100 micromol/l [Ca2+]) was significantly lower compared with that of the calmodulin-CaMK-2 association (K0.5 = 40 micromol/l [Ca2+], maximal at 1 mmol/l [Ca2+]). The data suggested that a transient Rab3A-calmodulin interaction might represent a means of directing calmodulin to the cytoplasmic face of a beta-granule, where it can be subsequently transferred for activation of other beta-granule-associated calmodulin-binding proteins as local [Ca2+]i rises to promote insulin exocytosis.

Dye-pair reporter systems for protein-peptide molecular interactions.

Modifying a linear peptide near each terminus with a fluorescent dye can make it able to signal its own binding to a protein. As originally described, the dye pair is composed of fluorescein and tetramethylrhodamine [Wei, A.-P., Blumenthal, D. K., and Herron, J. N. (1994) Anal. Chem. 66, 1500-1506]. This paper shows that it may also be two molecules of tetramethylrhodamine. In aqueous solution, mutual affinity of the dyes causes fluorescence-quenching contact between them. When the peptide is bound by an antibody or cleaved by a proteinase, or when acetonitrile is added, dye-to-dye contact decreases and fluorescence increases 3-15-fold. When five peptides of 4-20 amino acid residues were doubly modified with tetramethylrhodamine, each product had the absorption spectrum of a tetramethylrhodamine dimer. As the peptides were not known to have special conformational features, self-affinity of the dye appeared to be the main cause of dimerization. Disruption of the dye dimers by acetonitrile suggested that dimerization of the dye(s) in aqueous solution was largely an effect of hydrophobicity. Dye-tagged peptides were used in fluorometric assays for two peptide-protein interactions. First, a peptide from type II collagen recognized by a monoclonal antibody was derivatized with two different dye pairs. The monoclonal bound each modified peptide, disrupting dye-to-dye contact and increasing fluorescence up to 4-fold. Second, a phosphopeptide recognized by an SH2 domain was tagged with fluorescein and tetramethylrhodamine, and its binding to the SH2 domain was detected through fluorescence. Doubly dye-tagged peptides offer a direct, solution-phase assay for protein-peptide binding.

Spontaneous alpha-N-6-phosphogluconoylation of a "His tag" in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins.

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.

Cloning, expression, and type II collagenolytic activity of matrix metalloproteinase-13 from human osteoarthritic cartilage.

Proteolysis of triple-helical collagen is an important step in the progression toward irreversible tissue damage in osteoarthritis. Earlier work on the expression of enzymes in cartilage suggested that collagenase-1 (MMP-1) contributes to the process. Degenerate reverse transcription polymerase chain reaction experiments, Northern blot analysis, and direct immunodetection have now provided evidence that collagenase-3 (MMP-13), an enzyme recently cloned from human breast carcinoma, is expressed by chondrocytes in human osteoarthritic cartilage. Variable levels of MMP-13 and MMP-1 in cartilage was significantly induced at both the message and protein levels by interleukin-1 alpha. Recombinant MMP-13 cleaved type II collagen to give characteristic 3/4 and 1/4 fragments; however, MMP-13 turned over type II collagen at least 10 times faster than MMP-1. Experiments with intact type II collagen as well as a synthetic peptide suggested that MMP-13 cleaved type II collagen at the same bond as MMP-1, but this was then followed by a secondary cleavage that removed three amino acids from the 1/4 fragment amino terminus. The expression of MMP-13 in osteoarthritic cartilage and its activity against type II collagen suggest that the enzyme plays a significant role in cartilage collagen degradation, and must consequently form part of a complex target for proposed therapeutic interventions based on collagenase inhibition.