C-terminal Hydrophobic Core Research Articles

Energetic portrait of the amyloid beta nucleation transition state.

Amyloid protein aggregates are pathological hallmarks of more than fifty human diseases including the most common neurodegenerative disorders. The atomic structures of amyloid fibrils have now been determined, but the process by which soluble proteins nucleate to form amyloids remains poorly characterised and difficult to study, even though this is the key step to understand to prevent the formation and spread of aggregates. Here we use massively parallel combinatorial mutagenesis, a kinetic selection assay, and machine learning to reveal the transition state of the nucleation reaction of amyloid beta, the protein that aggregates in Alzheimer's disease. By quantifying the nucleation of >140,000 proteins we infer the changes in activation energy for all 798 amino acid substitutions in amyloid beta and the energetic couplings between >600 pairs of mutations. This unprecedented dataset provides the first comprehensive view of the energy landscape and the first large-scale measurement of energetic couplings for a protein transition state. The energy landscape reveals that the amyloid beta nucleation transition state contains a short structured C-terminal hydrophobic core with a subset of interactions similar to mature fibrils. This study demonstrates the feasibility of using mutation-selection-sequencing experiments to study transition states and identifies the key molecular species that initiates amyloid beta aggregation and, potentially, Alzheimer's disease.

Synthesis and Characterization of Propeller- and Parallel-Type Full-Length Amyloid β40 Trimer Models.

Oligomers of the amyloid β (Aβ) protein play a critical role in the pathogenesis of Alzheimer's disease. However, their heterogeneity and lability deter the identification of their tertiary structures and mechanisms of action. Aβ trimers and Aβ dimers may represent the smallest aggregation unit with cytotoxicity. Although propeller-type trimer models of E22P-Aβ40 tethered by an aromatic linker have recently been synthesized, they unexpectedly exhibited little cytotoxicity. To increase the flexibility of trimeric propeller-type models, we designed and synthesized trimer models with an alkyl linker, tert-butyltris-l-alanine (tButA), at position 36 or 38. In addition, we synthesized two parallel-type trimer models tethered at position 38 using alkyl linkers of different lengths, α,α-di-l-norvalyl-l-glycine (di-nV-Gly) and α,α-di-l-homonorleucyl-l-glycine (di-hnL-Gly), based on the previously reported toxic dimer model. The propeller-type E22P,V36tButA-Aβ40 trimer (4), which was designed to mimic the C-terminal anti-parallel β-sheet structures proposed by the structural analysis of 150 kDa oligomers of Aβ42, and the parallel-type E22P,G38di-nV-Gly-Aβ40 trimer (6) showed significant cytotoxicity against SH-SY5Y cells and aggregative ability to form protofibrillar species. In contrast, the E22P,G38tButA-Aβ40 trimer (5) and E22P,G38di-hnL-Gly-Aβ40 trimer (7) exhibited weak cytotoxicity, though they formed quasi-stable oligomers observed by ion mobility-mass spectrometry and native polyacrylamide gel electrophoresis. These results suggest that 4 and 6 could have some phase of the structure of toxic Aβ oligomers with a C-terminal hydrophobic core and that the conformation and/or aggregation process rather than the formation of stable oligomers contribute to the induction of cytotoxicity.

The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs, sequestering VP40 within IBs, reducing VP40–VLP egress, abolishing the incorporation of NC-like structures into VP40–VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

New diagnostic method for Alzheimer's disease based on the toxic conformation theory of amyloid β.

Recent investigations suggest that soluble oligomeric amyloid β (Aβ) species may be involved in early onset of Alzheimer's disease (AD). Using systematic proline replacement, solid-state NMR, and ESR, we identified a toxic turn at position 22 and 23 of Aβ42, the most potent neurotoxic Aβ species. Through radicalization, the toxic turn can induce formation of the C-terminal hydrophobic core to obtain putative Aβ42 dimers and trimers. Synthesized dimer and trimer models showed that the C-terminal hydrophobic core plays a critical role in the formation of high molecular weight oligomers with neurotoxicity. Accordingly, an anti-toxic turn antibody (24B3) that selectively recognizes a toxic dimer model of E22P-Aβ42 was developed. Sandwich enzyme-linked immunosorbent assay with 24B3 and 82E1 detected a significantly higher ratio of Aβ42 with a toxic turn to total Aβ42 in cerebrospinal fluid of AD patients compared with controls, suggesting that 24B3 could be useful for early onset of AD diagnosis.

Synthetic Models of Quasi-Stable Amyloid β40 Oligomers with Significant Neurotoxicity.

The formation of soluble oligomers of amyloid β42 and 40 (Aβ42, Aβ40) is the initial event in the pathogenesis of Alzheimer's disease (AD). Based on previous systematic proline replacement and solid-state NMR, we proposed a toxic dimer structure of Aβ42, a highly aggregative alloform, with a turn at positions 22 and 23, and a hydrophobic core in the C-terminal region. However, in addition to Aβ42, Aβ40 dimers can also contribute to AD progression because of the more abundance of Aβ40 monomer in biological fluids. Here, we describe the synthesis and characterization of three dimer models of the toxic-conformation constrained E22P-Aβ40 using l,l-2,6-diaminopimeric acid (DAP) or l,l-2,8-diaminoazelaic acid (DAZ) linker at position 30, which is incorporated into the intermolecular parallel β-sheet region, and DAP at position 38 in the C-terminal hydrophobic core. E22P-A30DAP-Aβ40 dimer (1) and E22P-A30DAZ-Aβ40 dimer (2) existed mainly in oligomeric states even after 2 weeks incubation without forming fibrils, unlike the corresponding monomer. Their neurotoxicity toward SH-SY5Y neuroblastoma cells was very weak. In contrast, E22P-G38DAP-Aβ40 dimer (3) formed β-sheet-rich oligomeric aggregates, and exhibited more potent neurotoxicity than the corresponding monomer. Ion mobility-mass spectrometry suggested that high molecular-weight oligomers (12-24-mer) of 3 form, but not for 1 and 2 after 4 h incubation. These findings indicate that formation of the hydrophobic core at the C-terminus, rather than intermolecular parallel β-sheet, triggers the formation of toxic Aβ oligomers. Compound 3 may be a suitable model for studying the etiology of Alzheimer's disease.

S-acylation anchors remorin proteins to the plasma membrane but does not primarily determine their localization in membrane microdomains.

Remorins are well-established marker proteins for plasma membrane microdomains. They specifically localize to the inner membrane leaflet despite an overall hydrophilic amino acid composition. Here, we determined amino acids and post-translational lipidations that are required for membrane association of remorin proteins. We used a combination of cell biological and biochemical approaches to localize remorin proteins and truncated variants of those in living cells and determined S-acylation on defined residues in these proteins. S-acylation of cysteine residues in a C-terminal hydrophobic core contributes to membrane association of most remorin proteins. While S-acylation patterns differ between members of this multi-gene family, initial membrane association is mediated by protein-protein or protein-lipid interactions. However, S-acylation is not a key determinant for the localization of remorins in membrane microdomains. Although remorins bind via a conserved mechanism to the plasma membrane, other membrane-resident proteins may be involved in the recruitment of remorins into membrane domains. S-acylation probably occurs after an initial targeting of the proteins to the plasma membrane and locks remorins in this compartment. As S-acylation is a reversible post-translational modification, stimulus-dependent intracellular trafficking of these proteins can be envisioned.

Stability and function of the Sec61 translocation complex depends on the Sss1p tail-anchor sequence

Sss1p, an essential component of the heterotrimeric Sec61 complex in the ER (endoplasmic reticulum), is a tail-anchored protein whose precise mechanism of action is largely unknown. Tail-anchored proteins are involved in many cellular processes and are characterized by a single transmembrane sequence at or near the C-terminus. The Sec61 complex is the molecular machine through which secretory and membrane proteins translocate into and across the ER membrane. To understand the function of the tail anchor of Sss1p, we introduced mutations into the tail-anchor sequence and analysed the resulting yeast phenotypes. Point mutations in the C-terminal hydrophobic core of the tail anchor of Sss1p were identified that allowed Sss1p assembly into Sec61 complexes, but resulted in diminished growth, defects in co- and post-translational translocation, inefficient ribosome binding to Sec61 complexes, reduction in the stability of both heterotrimeric Sec61 and heptameric Sec complexes and a complete breakdown of ER structure. The underlying defect caused by the mutations involves loss of a stabilizing function of the Sss1p tail-anchor sequence for both the heterotrimeric Sec61 and the heptameric Sec complexes. These results indicate that by stabilizing multiprotein membrane complexes, the hydrophobic core of a tail-anchor sequence can be more than a simple membrane anchor.

Why Is the C-terminus of Aβ(1−42) More Unfolded than That of Aβ(1−40)? Clues from Hydrophobic Interaction

Abeta(1-40) and Abeta(1-42) are the main forms of amyloid beta (Abeta) peptides in the brain of Alzheimer's patients; however, the latter possesses much stronger aggregation and deposition propensity than the former, which is partially attributed to the more unfolded C-terminus of Abeta(1-42) than that of Abeta(1-40). To explore the physical basis underlying the different dynamic behaviors of both Abeta peptides, parallel molecular dynamics (MD) simulations on Abeta(1-40) and Abeta(1-42) were performed to investigate their thermal unfolding processes. It is revealed that the addition of residues 41 and 42 in Abeta(1-42) disrupts the C-terminal hydrophobic core, which triggers the unraveling of the C-terminal helix of Abeta(1-42). This conclusion is supported by the MD simulation on the I41A mutant of Abeta(1-42), in which the C-terminal helix possesses relatively higher conformational stability than that of wild type Abeta(1-42) owing to the change in hydrophobic interaction patterns.

Hydrophobic Core around Tyrosine for Human Endothelin-1 Investigated by Photochemically Induced Dynamic Nuclear Polarization Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Human endothelin-1 (ET-1) is a potent cardiovascular bioactive peptide. Its activity is based on the C-terminal residues, e.g., Trp 21 in particular. Recently, we reported an NMR solution structure of ET-1, which has a C-terminal hydrophobic core around Tyr 13. This C-terminal conformation does not agree with a previously reported X-ray crystal structure. To clarify the discrepancy, we performed photo-CIDNP NMR in combination with MALDI-TOF MS. The photo-CIDNP results revealed that the Tyr 13 aromatic ring is concealed in a hydrophobic interaction. MALDI-TOF MS experiments showed this is an intramolecular interaction in monomeric form, which is also supported by sedimentation analysis and two-dimensional NMR cross-peak line shapes. Thus, we confirmed the intramolecular hydrophobic core around Tyr 13 in aqueous solution, which agrees with the solution structure. The C-terminal conformational discrepancy between the solution and crystal was caused by the intermolecular hydrogen bond between Tyr 13 of one molecule and Asp 8 of the other in a dimer-like formation of crystalline ET-1. On the other hand, we indicated that endothelin-3, another isoform of the endothelin, has an apparent self-association equilibrium under the same condition in which three tyrosines participate.

Loss of folylpoly-gamma-glutamate synthetase activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates in multiple human leukemia sublines.

We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates that target various folate-dependent enzymes. These include the dihydrofolate reductase (DHFR) inhibitors edatrexate, methotrexate and aminopterin, the thymidylate synthase (TS) inhibitors ZD1694 and GW1843, the glycinamide ribonucleotide formyltransferase (GARTF) inhibitor DDATHF as well as the multitargeted antifolate LY231514 inhibiting both TS, DHFR and GARTF. Fourteen antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired folylpoly-gamma-glutamate synthetase (FPGS) activity as these cell lines: 1) typically lost 90-99% of parental FPGS activity; 2) expressed 1.4-3.3-fold less FPGS mRNA (only 4 cell lines); 3) displayed up to 10(5)-fold resistance to polyglutamylation-dependent antifolates including ZD1694 and MTA; 4) retained sensitivity to polyglutamylation-independent antifolates including ZD9331 and PT523; 5) were up to 19-fold hypersensitive to the lipid-soluble antifolates trimetrexate and AG377; 6) had a normal or a small decrease in [(3)H]MTX transport; and 7) had a 2.1-8.3-fold decreased cellular folate pools and a consequently increased folate growth requirement. The remaining 3 antifolate-resistant sublines lost 94-97% of parental [(3)H]MTX transport and thus displayed a high level resistance to all hydrophilic antifolates. To screen for mutations in the hFPGS gene, we devised an RT-PCR single strand conformational polymorphism (SSCP) assay. RT-PCR-SSCP analysis and DNA sequencing showed that only a single FPGS-deficient subline harbored an FPGS mutation (Cys346Phe). Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within alpha-helix A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent antifolates but in the same time retain parental sensitivity or hypersensitivity to polyglutamylation-independent antifolates or lipophilic antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.

Guanidine hydrochloride-induced denaturation of the colicin E1 channel peptide: unfolding of local segments using genetically substituted tryptophan residues.

The soluble colicin E1 channel peptide has a roughly spherical, highly alpha-helical, compact structure. The structural unfolding properties of the colicin E1 channel peptide were analyzed using fluorescence techniques. The guanidine hydrochloride-induced unfolding pattern of the wild-type channel peptide was examined by monitoring intrinsic tryptophan fluorescence. Additionally, peptide unfolding was examined with the fluorophore, 1-anilinonaphthalene-8-sulfonic acid. In order to probe the unfolding of local segments, single-tryptophan channel peptides were constructed by site-directed mutagenesis. Shifts in fluorescence emission maxima of the single tryptophan residues were used to monitor site-specific unfolding events, in the presence of guanidine hydrochloride. The unfolding patterns reported by tryptophans in different regions of the peptide were diverse. The concentration of guanidine hydrochloride at the unfolding transition midpoint for each mutant peptide and the free energy of unfolding were calculated in order to estimate local segment stabilities. Also, secondary structure unfolding was monitored using circular dichroism spectroscopy. The results of unfolding analysis showed that the channel peptide's unfolding mechanism involves an intermediate structure stabilized by the C-terminal hydrophobic core of the peptide. Knowledge of the unfolding pattern of the soluble channel peptide will aid in the understanding of the secondary and tertiary structural interactions within the channel peptide and the mechanism of colicin E1 activation.