Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I: CONSTRAINTS AND FINE-TUNING IN THE EVOLUTION OF A PROTEIN FAMILY*

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His^B5 → Thr markedly destabilizes the hormone (ΔΔG_u 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr^B5 → His (residue 4) specifies a unique structure with native ¹H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His^B5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His^B5 does not significantly enhance thermodynamic stability (ΔΔG_u 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr^B5-insulin is decreased 5-fold, His^B5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr^B5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His^B5 in insulin highlights its critical role in insulin biosynthesis.     

Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo

Inhibition of the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca(2+) stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca(2+) content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca(2+) content by CaMKII. PLN(-/-) mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN(-/-) mice, indicating the involvement of a Ca(2+)-dependent pathway. AC3-I and AC3-C mice were bred into a PLN(-/-) background. Loss of PLN increased and equalized SR Ca(2+) content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend (P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca(2+) content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.     

Temperature and light requirements for growth of two diatom species (Bacillariophyceae) isolated from an Arctic macroalga

In the present study, two abundant epiphytic diatom taxa were isolated from the assimilation hairs of the brown macroalga Chordaria flagelliformis collected in the Arctic Kongsfjorden (Spitsbergen, Norway), established as unialgal cultures and their growth rates determined under controlled photon fluence rate and temperature conditions. Using morphological (light and scanning electron microscopy) and SSU rRNA gene data both isolates (ROS D99 and ROS D125) were identified as members of a Fragilaria–Synedropsis clade. The molecular data of ROS D99 and ROS D125 were not identical to any other published sequence. While ROS D99 has been identified as Fragilaria barbararum mainly due to the SEM characteristics, ROS D125 could not be definitely identified although morphological data speak for Fragilaria striatula. Both diatom species showed similar growth rates at all temperatures and photon fluence rates tested. They grew well between 0 and 15°C with optimum temperatures of 12–14°C, but did not survive 20°C. Therefore, compared to Antarctic diatoms both taxa from Kongsfjorden can be characterised as eurythermal organisms. Increasing photon fluence rates between 2 and 15 μmol m⁻² s⁻¹ were accompanied with an almost twofold increase in growth rates, but photon fluence rates >15 μmol m⁻² s⁻¹ did not further enhance growth pointing to low light requirements. From these data optimum, minimum and maximum photon fluence rates and temperatures for growth can be assessed indicating that both diatoms are well acclimated to the fluctuating environmental conditions in the Arctic habitat.     

Immunological characteristics correlating with clinical response to immunotherapy in patients with advanced metastatic melanoma

Current treatment options for advanced metastatic melanoma are limited to experimental regimen that provide poor survival outcomes. Immunotherapy is a promising alternative and we recently reported a clinical trial in which 6 out of 19 patients enrolled had objective clinical responses to a fully autologous melanoma/dendritic cell vaccine. The mechanism of the vaccine is not well understood, but we hypothesized that general immunocompetence may be a determinant of clinical response. We therefore examined the immune status of an expanded series of 21 patients who displayed varying clinical responses to the melanoma/dendritic cell vaccine. Immunocompetence was assessed using in vitro assays of lymphocyte function: survival, proliferation and cytokine responses to mitogen stimulation as well as T-cell receptor zeta expression and lymphocyte subset analysis. Although lymphocytes from patients mostly performed comparably to age-matched and sex-matched controls, in some assays we identified significant differences between complete clinical responders and other patients, both before and following vaccination. Surprisingly, before vaccination, only lymphocytes from clinical responder patients showed impaired in vitro survival. Following vaccination, T lymphocyte survival improved and cells recovered their ability to produce the Th1-associated cytokines TNF and IFN-gamma in response to anti-CD3 stimulation in vitro. No increase in Th1 cytokine production was observed in lymphocytes from patients who experienced partial clinical responses or progressive disease. We conclude that, before vaccination, patients who go on to have complete responses have immune characteristics suggestive of high cell turnover and low Th1-associated cytokine production, and that these can be reversed with vaccination. These results have potential implications for future immunotherapeutic strategies.