Effect Of C-terminal Truncation Research Articles

Structural Insights into α-Synuclein Fibril Polymorphism: Effects of Parkinson's Disease-Related C-Terminal Truncations

Lewy bodies, hallmarks of Parkinson's disease, contain C-terminally truncated (ΔC) α-synuclein (α-syn). Here, we report fibril structures of three N-terminally acetylated (Ac) α-syn constructs, Ac1–140, Ac1–122, and Ac1–103, solved by cryoelectron microscopy. Both ΔC-α-syn variants exhibited faster aggregation kinetics, and Ac1–103 fibrils efficiently seeded the full-length protein, highlighting their importance in pathogenesis. Interestingly, fibril helical twists increased upon the removal of C-terminal residues and can be propagated through cross-seeding. Compared to that of Ac1–140, increased electron densities were seen in the N-terminus of Ac1–103, whereas the C-terminus of Ac1–122 appeared more structured. In accord, the respective termini of ΔC-α-syn exhibited increased protease resistance. Despite similar amyloid core residues, distinctive features were seen for both Ac1–122 and Ac1–103. Particularly, Ac1–103 has the tightest packed core with an additional turn, likely attributable to conformational changes in the N-terminal region. These molecular differences offer insights into the effect of C-terminal truncations on α-syn fibril polymorphism.

Effect of C-Terminal Truncations on the Aggregation Propensity of Α- Synuclein - A Potential of Mean Force Study

α-Synuclein is an intrinsically disordered protein which has a prominent role in Parkinson’s disease (PD). Several familial variants of a-synuclein have been reported to associate with inherited PD. Several studies have highlighted the presence and association of the C-terminus of truncated forms of a-synuclein with Wild type α-synuclein (WT α- syn) at much higher level in patients with Lewy body diseases and they are found to decrease the reductive capabilities and potentially results in permanent oxidation of a-synuclein. To understand the impact of C-terminal truncations on the aggregation propensity of WT α-syn, the inter-molecular interactions between them are critical. Here we demonstrate the interactions between the WT α-syn and its three C-terminal truncations 95 (α-syn 95), 120 (α-syn 120) and 123 (α-syn 123), using Molecular Dynamics Simulations and Potential of Mean Force study (PMF). From the PMF study, we observed C-terminal truncation 120 (α-syn 120) to bind strongly to WT a-syn than the other truncated forms of a-synuclein. We also noticed number of hydrogen bonds to be larger, high geometrical shape complementarity score, larger number of interface residues and interface area for the hetero-dimer (WT α-syn-α-syn 120) than other hetero-dimers (WT α-syn - α-syn 95/123). We therefore can infer that among α-syn 95, α-syn 120 and α-syn 123, α-syn 120 to show more association with WT α-syn. This is because in α-syn 120, more amino acids have been removed in comparison to α-syn 123. In contrast, α-Syn 95 shows less interaction with WT α-syn because of the absence of the entire C-terminal region. Our findings suggest that more the truncation on the Cterminal region of α-synuclein more will the effect on its aggregation.

Effects of C-terminal truncation on autocatalytic processing of Bacillus licheniformis γ-glutamyl transpeptidase

The role of the C-terminal region of Bacillus licheniformis gamma-glutamyl transpeptidase (BlGGT) was investigated by deletion analysis. Seven C-terminally truncated BlGGTs lacking 581-585, 577-585, 576-585, 566-585, 558-585, 523-585, and 479-585 amino acids, respectively, were generated by site-directed mutagenesis. Deletion of the last nine amino acids had no appreciable effect on the autocatalytic processing of the enzyme, and the engineered protein was active towards the synthetic substrate L-gamma-glutamyl-p-nitroanilide. However, a further deletion to Val576 impaired the autocatalytic processing. In vitro maturation experiments showed that the truncated BlGGT precursors, pro-Delta(576-585), pro-Delta(566-585), and pro-Delta(558-585), could partially precede a time-dependent autocatalytic process to generate the L- and S-subunits, and these proteins showed a dramatic decrease in catalytic activity with respect to the wild-type enzyme. The parental enzyme (BlGGT-4aa) and BlGGT were unfolded biphasically by guanidine hydrochloride (GdnCl), but Delta(577-585), Delta(576-585), Delta(566-585), Delta(558-585), Delta(523-585), and Delta(479-585) followed a monophasic unfolding process and showed a sequential reduction in the GdnCl concentration corresponding to half effect and DeltaG(0) for the unfolding. BlGGT-4aa and BlGGT sedimented at ~4.85 S and had a heterodimeric structure of approximately 65.23 kDa in solution, and this structure was conserved in all of the truncated proteins. The frictional ratio (f/f(o)) of BlGGT-4aa, BlGGT, Delta(581-585), and Delta(577-585) was 1.58, 1.57, 1.46, and 1.39, respectively, whereas the remaining enzymes existed exclusively as precursor form with a ratio of less than 1.18. Taken together, these results provide direct evidence for the functional role of the C-terminal region in the autocatalytic processing of BlGGT.

Intracellular lipid droplet targeting by apolipoprotein A-V requires the carboxyl-terminal segment

The expression of apolipoprotein A-V (apoA-V) in hepatoma cells results in homing of this protein to intracellular lipid droplets. When hepatoma cells transfected with a full-length apoA-V-green fluorescent protein fusion protein were cultured in medium that was not supplemented with oleic acid (OA), intracellular lipid droplet size and number were reduced compared with those of cells supplemented with OA. Confocal microscopy studies revealed that apoA-V associates with lipid droplets under both conditions. To define the structural requirements for apoA-V lipid droplet association, hepatoma cells were transfected with a series of C-terminal truncated apoA-V variants. Confocal microscopy analysis revealed that, in a manner similar to mature full-length apoA-V (343 amino acids), truncation variants apoA-V(1-292), apoA-V(1-237), and apoA-V(1-191) associated with lipid droplets, while apoA-V(1-146) did not. Western blot analysis of the relative abundance of apoA-V in cell lysates versus conditioned medium indicated that apoA-V variants associated with lipid droplets were poorly secreted while apoA-V(1-146) was efficiently secreted. Ultracentrifugation of conditioned medium revealed that, unlike full-length apoA-V, which associates with lipoproteins, apoA-V(1-146) was present solely in the lipoprotein-deficient fraction. Deletion of the N-terminal signal peptide from apoA-V resulted in an inability of the protein to be secreted into the medium, although it associated with lipid droplets. Taken together, these data suggest that the C terminus of apoA-V is essential for lipid droplet association in transfected hepatoma cells and lipoprotein association in conditioned medium while the signal peptide is required for extracellular trafficking of this protein.

Effects of C-terminal Truncations on Trafficking of the Yeast Plasma Membrane H+-ATPase

Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.

Thermoregulatory Activity of Dermorphin Fragments

We studied the effect of C-terminal truncation of the dermorphin (DM) molecule and analogs of its N-terminal tetrapeptide, [DOrn2]-DM(1-4), [DArg2]-DM(1-4), [DAla4]-DM(1-4), [DArg2, DAla4]-DM(1-4), Arg-DM(1-4), Arg-[DArg2]-DM(1-4), Arg-[DAla4]-DM(1-4), and Arg-[DArg2, DAla4]-DM(1-4), on the functional status of the thermoregulation system in rats at different ambient temperatures. For the first time, we demonstrate that the N-terminal tetrapeptide is the minimal fragment with the hypothermic effect. Only the N-terminal octapeptide exerted the vasomotor effect. Amino acid substitutions in the tetrapeptide affected its hypothermic effect. [DArg2]-DM(1-4) and [DArg2, DAla4]-DM(1-4) had the greatest effect. Addition of Arg to the N-terminus of DM(1-4) analogs changed their thermoregulatory activity. The greatest thermoregulatory effect was observed for Arg-[DArg2]-DM(1-4) and Arg-[DArg2, DAla4]-DM(1-4).

Folding of an abridged beta-lactamase.

The effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small beta-lactamase (ES-betaL) have been measured. ES-betaL lacking 19 residues (ES-betaL(C)(Delta)(19)) has no enzymic activity. Deletion of the last 14 residues produces ES-betaL(C)(Delta)(14), which is 0.1% active. The enzyme lacking nine residues (ES-betaL(C)(Delta)(9)) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-betaL(C)(Delta)(9) folds properly, it does so 4 orders of magnitude slower than ES-betaL, making possible the isolation and characterization of a compact intermediate state (I(P) ES-betaL(C)(Delta)(9)). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between I(P) ES-betaL(C)(Delta)(9) and other intermediate slow folding. Residues removed in ES-betaL(C)(Delta)(9) and ES-betaL(C)(Delta)(14) are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.

Effect of C-terminal truncations on MLK7 catalytic activity and JNK activation

Mixed lineage kinase 7 (MLK7) is a MAPKKK with enriched expression in heart and skeletal muscle that functions to activate JNK and p38. The MLKs have several conserved domains, including a leucine zipper that in other family members mediates oligomerization critical for catalytic activity and JNK activation. Nested C-terminal deletion mutants of MLK7 from 436 to 286 as well as a mutant lacking only the leucine zipper (delLZ) were generated to determine the role of these domains in catalytic activity and JNK activation. Specific activity of MLK7366 was 75% full length while 436, 322, and delLZ retained approximately 25% and 286, 4% of the full-length catalytic function, demonstrating that the leucine zipper, while not absolutely necessary for catalytic activity, is required to reach full catalytic function of the enzyme. Co-transfection studies of JNK with the MLK7 mutants demonstrated full JNK activation with MLK7, 436, and delLZ, marginal activation for 1–400 or 1–366, and no activation for 1–322, demonstrating that the leucine zipper is not required for JNK activation and that sequence contained in C-terminal residue 322–436 is necessary for full pathway activation by MLK7.

Hydrodynamic properties and quaternary structure of the 90 kDa heat-shock protein: effects of divalent cations.

The 90 kDa heat-shock protein (Hsp90) is one of the major stress proteins whose overall structure remains unknown. In this study, we investigated the influence of divalent cations Mg(2+) and Ca(2+) on the hydrodynamic properties and quaternary structure of Hsp90. Using analytical ultracentrifugation, size-exclusion chromatography, and polyacrylamide gel electrophoresis, we showed that native Hsp90 was mostly dimeric. The Hsp90 dimer had a sedimentation coefficient, s(w,20) degrees, of 6.10 +/- 0.03 S, which slightly deviated from the hydrodynamics of a globular protein. Using chemical cross-linking and analytical ultracentrifugation, we showed that Mg(2+) and Ca(2+) induced a tertiary conformational change of Hsp90, leading to a self-association process. In the presence of divalent cations, Hsp90 existed as a mixture of monomers, dimers, and tetramers at equilibrium. Finally, to identify Hsp90 domains involved in this divalent cation-dependent self-association, we studied the oligomerization state of the N-terminal (positions 1-221) of Hsp90, the influence of an N-terminal specific ligand, geldanamycin (GA), and the effect of C-terminal truncation on the ability of Hsp90 to oligomerize in the presence of divalent cations. We previously showed that GA inhibits Hsp90 heat-induced oligomerization [Garnier, C., Protasevich, I., Gilli, R., Tsvetkov, P., Lobachov, V., Peyrot, V., Briand, C., and Makarov, A. (1998) Biochem. Biophys. Res. Commun. 249, 197-201], but now we observed that GA does not influence divalent cation-dependent oligomerization of Hsp90, suggesting another mechanism. This mechanism involved the C-terminal part of the protein since C-terminally truncated Hsp90 did not oligomerize in the presence of divalent cations.

Chemically prepared hevein domains: effect of C-terminal truncation and the mutagenesis of aromatic residues on the affinity for chitin.

Chemically prepared hevein domains (HDs), N-terminal domain of an antifungal protein from Nicotiana tabacum (CBP20-N) and an antimicrobial peptide from Amaranthus caudatus (Ac-AMP2), were examined for their affinity for chitin, a beta-1,4-linked polymer of N-acetylglucosamine. An intact binding domain, CBP20-N, showed a higher affinity than a C-terminal truncated domain, Ac-AMP2. The formation of a pyroglutamate residue from N-terminal Gln of CBP20-N increased the affinity. The single replacement of any aromatic residue of Ac-AMP2 with Ala resulted in a significant reduction in affinity, suggesting the importance of the complete set of three aromatic residues in the ligand binding site. The mutations of Phe18 of Ac-AMP2 to the residues with larger aromatic rings, i.e. Trp, beta-(1-naphthyl)alanine or beta-(2-naphthyl)alanine, enhanced the affinity, whereas the mutation of Tyr20 to Trp reduced the affinity. The affinity of an HD for chitin might be improved by adjusting the size and substituent group of stacking aromatic rings.