Identification of the allosteric regulatory site of insulysin

Background

Insulin degrading enzyme (IDE) is responsible for the metabolism of insulin and plays a role in clearance of the Aβ peptide associated with Alzheimer''s disease. Unlike most proteolytic enzymes, IDE, which consists of four structurally related domains and exists primarily as a dimer, exhibits allosteric kinetics, being activated by both small substrate peptides and polyphosphates such as ATP.

Principal Findings

The crystal structure of a catalytically compromised mutant of IDE has electron density for peptide ligands bound at the active site in domain 1 and a distal site in domain 2. Mutating residues in the distal site eliminates allosteric kinetics and activation by a small peptide, as well as greatly reducing activation by ATP, demonstrating that this site plays a key role in allostery. Comparison of the peptide bound IDE structure (using a low activity E111F IDE mutant) with unliganded wild type IDE shows a change in the interface between two halves of the clamshell-like molecule, which may enhance enzyme activity by altering the equilibrium between closed and open conformations. In addition, changes in the dimer interface suggest a basis for communication between subunits.

Conclusions/Significance

Our findings indicate that a region remote from the active site mediates allosteric activation of insulysin by peptides. Activation may involve a small conformational change that weakens the interface between two halves of the enzyme.     

Distribution of [3H]dexamethasone in rat subcutaneous tissue after delivery from osmotic pumps

Inflammation surrounding implantable glucose sensors may be controlled through local release of dexamethasone at the site of implantation. In the present study, we evaluated the distribution of dexamethasone in rat subcutaneous tissue during the first 2.5 days after local release. Osmotic pumps containing [3H]dexamethasone were implanted into the subcutaneous tissue of rats. Digital autoradiography was used to measure the distribution of the [3H]dexamethasone within the subcutaneous tissue at 6, 24, and 60 h after implantation. Measured concentration profiles, near the catheter tip through which the agent was released, were compared to mathematical models of drug diffusion and elimination. The results demonstrate that the majority of the [3H]dexamethasone delivered into the subcutaneous tissue was found within a 3 mm region surrounding the catheter tip. There was good agreement between the experimental data and the mathematical model. The diffusion coefficient for dexamethasone in subcutaneous tissue was found to be D = 4.11 +/- 1.77 x 10(-10) m2/s, and the elimination rate constant was found to be k = 3.65 +/- 2.24 x 10(-5) s(-1). The diffusion coefficient and elimination rate constants for dexamethasone in subcutaneous tissue have not been previously reported. The use of a mathematical model may be useful in predicting the effectiveness of local delivery of dexamethasone around implantable glucose sensors.     

Transduction of human dendritic cells with a recombinant modified vaccinia Ankara virus encoding MUC1 and IL-2

The epithelial mucin MUC1 is considered an opportune target antigen for cancer immunotherapy, as it is over-expressed and exhibits aberrant glycosylation in malignant cells. Because dendritic cells (DC) are powerful initiators of immune responses, efforts have focused on tumor antigen-bearing DC as potent cancer vaccines. In this study we have characterized the transduction of monocyte-derived DC with a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] encoding human MUC1 and the immunostimulatory cytokine IL-2. Analysis of transduced DC cultures generated from a number of donors revealed MUC1 expression in the range of 27-54% of the cells and a co-regulated secretion of bioactive IL-2. As shown by FACS analysis with MUCI-specific antibodies, the MVA-MUC1/IL-2-transduced DC predominantly expressed the fully processed glycoform of MUC1, typical of that displayed by normal epithelia. Over a 3-day period after transduction, transgene expression declined concurrent with an increase in MVA-induced cytopathic effects. The transduced DC stimulated allogeneic lymphocyte proliferation, indicating that DC immunostimulatory function is not impaired by vector transduction. In the presence of IL-2, MVA-transduced DC were able to enhance autologous lymphocyte proliferation. Also, vector expression was analyzed in DC cultures treated with TNF-alpha, a known DC maturation factor. As indicated by the up-regulation of several DC maturation markers, neither virus infection nor transgene expression influenced the maturation capacity of the cells. The MVA-MUC1/IL-2 vector effectively transduced both immature and TNF-alpha-matured DC. Overall, our results are encouraging for the clinical application of MVA-MUC1/IL-2-transduced DC.     

Chemoimmunotherapy of advanced breast cancer: prolongation of remission and survival with BCG.

Forty-five patients with disseminated breast cancer were given a trial of combination chemotherapy consisting of fluorouracil, adriamycin, and cyclophosphamide (FAC) and immunotherapy with BCG given by scarification. The results were compared with those in a comparable group of 44 patients treated with FAC alone immediately before the chemoimmunotherapy study. The remission rates (73% and 76% for FAC and FAC-BCG respectively) were similar in both studies. The durations of remission for patients on FAC-BCG (medium 12 months) were longer than remissions achieved for patients given FAC alone (median 8 months) (P = 0.068). The most notable effect of BCG was on survival. Thus 21 out of 34 patients achieving remission on FAC-BCG were alive at the time of the last follow-up examination (median over 22 months) compared with 11 out of 32 patients achieving remission on FAC (median 15 months) (P = 0.01). Twenty-six of the 45 patients given FAC-BCG were alive at the time of the last follow-up examination (median over 22 months) compared with 12 of the 44 patients given FAC (median 15 months) (P = 0.005). Although the apparent benefit of BCG could be explained by a maldistribution of some prognostic factors, the data suggest that further trial of chemoimmunotherapy of breast cancer should be carried out.     

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

Immune Function in Multiple Myeloma: Impaired Responsiveness to Keyhole Limpet Hemocyanin

Twenty-three patients with multiple myeloma, four patients with treated localized plasmacytoma and 14 normal subjects were immunized with keyhole limpet hemocyanin (KLH). When compared to the normal subjects, the myeloma patients showed a prolonged induction time for IgM antibody formation, a more rapid switch from IgM to IgG production and a decline in the titre of total antibody produced. In vitro lymphocyte responses to KLH following immunization were reduced in the myeloma group and tended to decline with time in a manner similar to the serum antibody concentration. Most of the myeloma patients tested developed delayed hypersensitivity skin reactions to KLH, but these reactions were smaller than those of the control subjects. The patients with myeloma had also reduced in vitro lymphocyte responses to streptolysin-O and vaccinia. Immune function of the plasmacytoma patients was similar to that of the control subjects.Both humoral and cellular immunity in response to a newly encountered antigen, KLH, is impaired in patients with multiple myeloma.