Deletion Of N-terminal Residues Research Articles

∆nFGF1 Protects β-Cells against High Glucose-Induced Apoptosis via the AMPK/SIRT1/PGC-1α Axis.

Long-term exposure to high glucose leads to β-cell dysfunction and death. Fibroblast growth factor 1 (FGF1) has emerged as a promising diabetes treatment, but its pharmaceutical role and mechanism against glucolipotoxicity-induced β-cell dysfunction remain uncharacterized. Wild-type FGF1 (FGF1WT) may exhibit in vivo mitogenicity, but deletion of N-terminal residues 1-27 gives a nonmitogenic variant, ∆nFGF1, that does not promote cell proliferation and still retains the metabolic activity of FGF1WT. To investigate the roles of ∆nFGF1 on glucose regulation and potential islet β-cell dysfunction, db/db mice were used as a model of type 2 diabetes. The results showed that insulin secretion and apoptosis of islet β-cells were dramatically improved in ∆nFGF1-treated db/db mice. To further test the effects of ∆nFGF1 treatment, pancreatic β-cell (MIN6) cells were exposed to a mixture of palmitic acid (PA) and high glucose (HG) to mimic glucolipotoxic conditions in vitro. Treatment with ∆nFGF1 significantly inhibited glucolipotoxicity-induced apoptosis. Mechanistically, ∆nFGF1 exerts a protective effect on β-cells via activation of the AMPK/SIRT1/PGC-1α signaling pathway. These findings demonstrate that ∆nFGF1 protects pancreatic β-cells against glucolipotoxicity-induced dysfunction and apoptosis.

ATP-dependent membrane remodeling links EHD1 functions to endocytic recycling

Endocytic and recycling pathways generate cargo-laden transport carriers by membrane fission. Classical dynamins, which generate transport carriers during endocytosis, constrict and cause fission of membrane tubes in response to GTP hydrolysis. Relatively, less is known about the ATP-binding Eps15-homology domain-containing protein1 (EHD1), a dynamin family member that functions at the endocytic-recycling compartment. Here, we show using cross complementation assays in C. elegans that EHD1’s membrane binding and ATP hydrolysis activities are necessary for endocytic recycling. Further, we show that ATP-bound EHD1 forms membrane-active scaffolds that bulge tubular model membranes. ATP hydrolysis promotes scaffold self-assembly, causing the bulge to extend and thin down intermediate regions on the tube. On tubes below 25 nm in radius, such thinning leads to scission. Molecular dynamics simulations corroborate this scission pathway. Deletion of N-terminal residues causes defects in stable scaffolding, scission and endocytic recycling. Thus, ATP hydrolysis-dependent membrane remodeling links EHD1 functions to endocytic recycling.

Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity.

Functional Dissection of N -Acetylglutamate Synthase (ArgA) of Pseudomonas aeruginosa and Restoration of Its Ancestral N -Acetylglutamate Kinase Activity

In many microorganisms, the first step of arginine biosynthesis is catalyzed by the classical N-acetylglutamate synthase (NAGS), an enzyme composed of N-terminal amino acid kinase (AAK) and C-terminal histone acetyltransferase (GNAT) domains that bind the feedback inhibitor arginine and the substrates, respectively. In NAGS, three AAK domain dimers are interlinked by their N-terminal helices, conforming a hexameric ring, whereas each GNAT domain sits on the AAK domain of an adjacent dimer. The arginine inhibition of Pseudomonas aeruginosa NAGS was strongly hampered, abolished, or even reverted to modest activation by changes in the length/sequence of the short linker connecting both domains, supporting a crucial role of this linker in arginine regulation. Linker cleavage or recombinant domain production allowed the isolation of each NAGS domain. The AAK domain was hexameric and inactive, whereas the GNAT domain was monomeric/dimeric and catalytically active although with ∼50-fold-increased and ∼3-fold-decreased K(m)(glutamate) and k(cat) values, respectively, with arginine not influencing its activity. The deletion of N-terminal residues 1 to 12 dissociated NAGS into active dimers, catalyzing the reaction with substrate kinetics and arginine insensitivity identical to those for the GNAT domain. Therefore, the interaction between the AAK and GNAT domains from different dimers modulates GNAT domain activity, whereas the hexameric architecture appears to be essential for arginine inhibition. We proved the closeness of the AAK domains of NAGS and N-acetylglutamate kinase (NAGK), the enzyme that catalyzes the next arginine biosynthesis step, shedding light on the origin of classical NAGS, by showing that a double mutation (M26K L240K) in the isolated NAGS AAK domain elicited NAGK activity.

Oligomerization of BH4-truncated Bcl-x L in solution

BH4 domain is critical for the anti-apoptotic functions of Bcl-2 and Bcl-x L and their binding abilities with other members of the Bcl-2 family. The cleavage of the BH4 domain in Bcl-x L and Bcl-2 by caspase 1 or 3 converts the anti-apoptotic Bcl-x L and Bcl-2 into pro-apoptotic proteins that potently induce apoptosis. Herein, we report that recombinant Bcl-x L proteins without N-terminal 61 residues, His 6-NΔ61-Bcl-x L-CΔ21 and NΔ61-Bcl-x L-CΔ21, form oligomers in solution, whereas Bcl-x L-CΔ21 exists as a monomer. The oligomerization of the truncated proteins is independent of protein–lipid interaction, protein concentration or the ion strength of the solution. Circular dichroism spectrum shows a significant decrease in the content of α-helices upon deletion of N-terminal residues. NΔ61-Bcl-x L-CΔ21 also loses its heterodimerization capability with the BH3 peptide derived from Bak. This newly acquired property might be linked to its ability to induce apoptosis in cells.

Inhibition of cardiac L-type calcium channels by protein kinase C phosphorylation of two sites in the N-terminal domain

We have investigated the mechanism underlying the modulation of the cardiac L-type Ca(2+) current by protein kinase C (PKC). Using the patch-clamp technique, we found that PKC activation by 4-alpha-phorbol 12-myristate 13-acetate (PMA) or rac-1-oleyl-2-acetylglycerol (OAG) caused a substantial reduction in Ba(2+) current through Ca(v)1.2 channels composed of alpha(1)1.2, beta(1b), and alpha(2)delta(1) subunits expressed in tsA-201 cells. In contrast, Ba(2+) current through a cloned brain isoform of the Ca(v)1.2 channel (rbC-II) was unaffected by PKC activation. Two potential sites of PKC phosphorylation are present at positions 27 and 31 in the cardiac form of Ca(v)1.2, but not in the brain form. Deletion of N-terminal residues 2-46 prevented PKC inhibition. Conversion of the threonines at positions 27 and 31 to alanine also abolished the PKC sensitivity of Ca(v)1.2. Mutant Ca(v)1.2 channels in which the threonines were converted singly to alanines were also insensitive to PKC modulation, suggesting that phosphorylation of both residues is required for PKC-dependent modulation. Consistent with this, mutating each of the threonines individually to aspartate in separate mutants restored the PKC sensitivity of Ca(v)1.2, indicating that a change in net charge by phosphorylation of both sites is responsible for inhibition. Our results define the molecular basis for inhibition of cardiac Ca(v)1.2 channels by the PKC pathway.

Minimal Ligand Analysis of Gastrin‐Releasing Peptide: Receptor Binding and Mitogenesis

Gastrin releasing peptide (GRP) is a peptide hormone containing 27 amino acids which is structurally analogous to the amphibian peptide bombesin. GRP serves a variety of physiological functions and has been implicated in the pathophysiology of small cell lung cancer. Previous work has demonstrated that the modified C terminus of GRP, N-acetyl-GRP-20-27, exerts full agonist activity in a variety of assay systems. However, no systematic comparison of binding of GRP fragments to its receptor and mitogenic potency has been reported. To investigate whether smaller GRP fragments could bind to the GRP receptor without stimulating mitogenesis, we performed binding inhibition and thymidine uptake assays with Swiss 3T3 fibroblasts. These studies were facilitated by the development of a novel tritiated GRP-based radioligand, [3H-Phe15] GRP-15-27, which exhibits enhanced chemical stability compared to iodinated GRP derivatives. We examined a series of C-terminal GRP fragments, from the pentapeptide to the octapeptide, with both N-acetyl and free amine moieties at the N terminus. N-Acetylated derivatives were more potent than their primary amine counterparts in both assays. Deletion of N-terminal residues from GRP-20-27 resulted in significant loss of potency in both assays: the EC50 values of N-acetyl-GRP-21-27 were 10(2)-fold higher than N-acetyl-GRP-20-27, those of N-acetyl-GRP-22-27 were 10(4)-fold higher, and N-acetyl-GRP-23-27 showed minimal activity at concentrations below 100 microM. These results suggest that 1) both His20 and Trp21 play an important role in binding of GRP to the receptor, and 2) for this series of N-terminal deletions, binding to the receptor and mitogenic activity are tightly coupled.

Minimal ligand analysis of gastrin releasing peptide. Receptor binding and mitogenesis.

Gastrin releasing peptide (GRP) is a peptide hormone containing 27 amino acids which is structurally analogous to the amphibian peptide bombesin. GRP serves a variety of physiological functions and has been implicated in the pathophysiology of small cell lung cancer. Previous work has demonstrated that the modified C terminus of GRP, N-acetyl-GRP-20-27, exerts full agonist activity in a variety of assay systems. However, no systematic comparison of binding of GRP fragments to its receptor and mitogenic potency has been reported. To investigate whether smaller GRP fragments could bind to the GRP receptor without stimulating mitogenesis, we performed binding inhibition and thymidine uptake assays with Swiss 3T3 fibroblasts. These studies were facilitated by the development of a novel tritiated GRP-based radioligand, [3H-Phe15] GRP-15-27, which exhibits enhanced chemical stability compared to iodinated GRP derivatives. We examined a series of C-terminal GRP fragments, from the pentapeptide to the octapeptide, with both N-acetyl and free amine moieties at the N terminus. N-Acetylated derivatives were more potent than their primary amine counterparts in both assays. Deletion of N-terminal residues from GRP-20-27 resulted in significant loss of potency in both assays: the EC50 values of N-acetyl-GRP-21-27 were 10(2)-fold higher than N-acetyl-GRP-20-27, those of N-acetyl-GRP-22-27 were 10(4)-fold higher, and N-acetyl-GRP-23-27 showed minimal activity at concentrations below 100 microM. These results suggest that 1) both His20 and Trp21 play an important role in binding of GRP to the receptor, and 2) for this series of N-terminal deletions, binding to the receptor and mitogenic activity are tightly coupled.