Bertil Macao

Stabilization of neurotoxic Alzheimer amyloid-β oligomers by protein engineering

Soluble oligomeric aggregates of the amyloid-β peptide (Aβ) have been implicated in the pathogenesis of Alzheimer’s disease (AD). Although the conformation adopted by Aβ within these aggregates is not known, a β-hairpin conformation is known to be accessible to monomeric Aβ. Here we show that this β-hairpin is a building block of toxic Aβ oligomers by engineering a double-cysteine mutant (called Aβcc) in which the β-hairpin is stabilized by an intramolecular disulfide bond. Aβ40cc and Aβ42cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aβ aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Aβcc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that β-sheet-containing Aβ42cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Aβ42, in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Aβ oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Aβ42 is more toxic than the shorter Aβ40, and why certain inherited mutations are linked to protofibril formation and early-onset AD.

Autoproteolysis coupled to protein folding in the SEA domain of the membrane-bound MUC1 mucin

The single cell layer of the lungs and the gastrointestinal tract is protected by the mucus formed by large glycoproteins called mucins. Transmembrane mucins typically contain 110-residue SEA domains located next to the membrane. These domains undergo post-translational cleavage between glycine and serine in a characteristic GSVVV sequence, but the two peptides remain tightly associated. We show that the SEA domain of the human MUC1 transmembrane mucin undergoes a novel type of autoproteolysis, which is catalyzed by conformational stress and the conserved serine hydroxyl. We propose that self-cleaving SEA domains have evolved to dissociate as a result of mechanical rather than chemical stress at the apical cell membrane and that this protects epithelial cells from rupture. We further suggest that the cell can register mechanical shear at the mucosal surface if the dissociation is signaled via loss of a SEA-binding protein.

Sequestration of the Aβ Peptide Prevents Toxicity and Promotes Degradation In Vivo

An engineered protein prevents aggregation of the Aβ peptide and facilitates clearance of Aβ from the brain in a fruit fly model of Alzheimers disease.

Protein autoproteolysis: conformational strain linked to the rate of peptide cleavage by the pH dependence of the N --> O acyl shift reaction.

Nucleophilic attack by a side chain nucleophile on the adjacent peptide bond followed by N --> O or N --> S acyl shift is the primary step in protein autoproteolysis. Precursor structures of autoproteolytic proteins reveal strained (or twisted) amides at the site of cleavage, and we previously showed that SEA domain autoproteolysis involves substrate destabilization by approximately 7 kcal/mol. However, the precise chemical mechanism by which conformational energy is converted into reaction rate acceleration has not been understood. Here we show that the pH dependence of autoproteolysis in a slow-cleaving mutant (1G) of the MUC1 SEA domain is consistent with a mechanism in which N --> O acyl shift proceeds after initial protonation of the amide nitrogen. Unstrained amides have pK(a) values of 0 with protonation on the oxygen, and autoproteolysis is therefore immeasurably slow at neutral pH. However, conformational strain forces the peptide nitrogen into a pyramidal conformation with a significantly increased pK(a) for protonation. We find that pK(a) values of approximately 4 and approximately 6, as in model compounds of twisted amides, reproduce the rate of autoproteolysis in the 1G and wild-type SEA domains, respectively. A mechanism involving strain, nitrogen protonation, and N --> O shift is also supported by quantum-chemical calculations. Such a reaction therefore constitutes an alternative to peptide cleavage that is utilized in autoproteolysis, as opposed to a classical mechanism involving a structurally conserved active site with a catalytic triad and an oxyanion hole, which are not present at the SEA domain cleavage site.

Recombinant amyloid beta-peptide production by coexpression with an affibody ligand.

BackgroundOligomeric and fibrillar aggregates of the amyloid β-peptide (Aβ) have been implicated in the pathogenesis of Alzheimers disease (AD). The characterization of Aβ assemblies is essential for the elucidation of the mechanisms of Aβ neurotoxicity, but requires large quantities of pure peptide. Here we describe a novel approach to the recombinant production of Aβ. The method is based on the coexpression of the affibody protein ZAβ3, a selected affinity ligand derived from the Z domain three-helix bundle scaffold. ZAβ3 binds to the amyloidogenic central and C-terminal part of Aβ with nanomolar affinity and consequently inhibits aggregation.ResultsCoexpression of ZAβ3 affords the overexpression of both major Aβ isoforms, Aβ(1–40) and Aβ(1–42), yielding 4 or 3 mg, respectively, of pure 15N-labeled peptide per liter of culture. The method does not rely on a protein-fusion or -tag and thus does not require a cleavage reaction. The purified peptides were characterized by NMR, circular dichroism, SDS-PAGE and size exclusion chromatography, and their aggregation propensities were assessed by thioflavin T fluorescence and electron microscopy. The data coincide with those reported previously for monomeric, largely unstructured Aβ. ZAβ3 coexpression moreover permits the recombinant production of Aβ(1–42) carrying the Arctic (E22G) mutation, which causes early onset familial AD. Aβ(1–42)E22G is obtained in predominantly monomeric form and suitable, e.g., for NMR studies.ConclusionThe coexpression of an engineered aggregation-inhibiting binding protein offers a novel route to the recombinant production of amyloidogenic Aβ peptides that can be advantageously employed to study the molecular basis of AD. The presented expression system is the first for which expression and purification of the aggregation-prone Arctic variant (E22G) of Aβ(1–42) is reported.

Ffh and FtsY in a Mycoplasma mycoides signal-recognition particle pathway: SRP RNA and M domain of Ffh are not required for stimulation of GTPase activity in vitro.

Mycoplasma mycoides contains a signal‐recognition particle (SRP) composed of an RNA molecule and an SRP54 homologue (Ffh). We have now identified a mycoplasma homologue to the α subunit of the mammalian SRP receptor and Escherichia coli FtsY. The protein (MmFtsY) was expressed in E. coli and purified to homogeneity. MmFtsY has a weak intrinsic GTPase activity but GTP hydrolysis was markedly stimulated when it was combined with mycoplasma Ffh (MmFfh) and SRP RNA. Also, in the absence of SRP RNA GTPase activity was significantly enhanced. Furthermore, GTP hydrolysis was stimulated when MmFtsY was combined with the N‐terminal GTPase domain (N+G) of MmFfh. These findings indicate that basic features of the GTPase activation mechanism are independent of the C‐terminal M domain of the MmFfh protein. We propose that the activation is mediated to a large extent by contacts between the GTPase domains of the mycoplasma Ffh and FtsY proteins and that the contribution of the M domain and SRP RNA in the activation mechanism is mainly for modifying the conformation of the MmFfh GTPase domain.

The exonuclease activity of DNA polymerase γ is required for ligation during mitochondrial DNA replication

Mitochondrial DNA (mtDNA) polymerase γ (POLγ) harbours a 3′–5′ exonuclease proofreading activity. Here we demonstrate that this activity is required for the creation of ligatable ends during mtDNA replication. Exonuclease-deficient POLγ fails to pause on reaching a downstream 5′-end. Instead, the enzyme continues to polymerize into double-stranded DNA, creating an unligatable 5′-flap. Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. In mice, inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes. These mutator mice also contain high levels of truncated, linear fragments of mtDNA. We demonstrate that the formation of these fragments is due to impaired ligation, causing nicks near the origin of heavy-strand DNA replication. In the subsequent round of replication, the nicks lead to double-strand breaks and linear fragment formation.

Sequence-specific stalling of DNA polymerase γ and the effects of mutations causing progressive ophthalmoplegia

A large number of mutations in the gene encoding the catalytic subunit of mitochondrial DNA polymerase γ (POLγA) cause human disease. The Y955C mutation is common and leads to a dominant disease with progressive external ophthalmoplegia and other symptoms. The biochemical effect of the Y955C mutation has been extensively studied and it has been reported to lower enzyme processivity due to decreased capacity to utilize dNTPs. However, it is unclear why this biochemical defect leads to a dominant disease. Consistent with previous reports, we show here that the POLγA:Y955C enzyme only synthesizes short DNA products at dNTP concentrations that are sufficient for proper function of wild-type POLγA. In addition, we find that this phenotype is overcome by increasing the dNTP concentration, e.g. dATP. At low dATP concentrations, the POLγA:Y955C enzyme stalls at dATP insertion sites and instead enters a polymerase/exonuclease idling mode. The POLγA:Y955C enzyme will compete with wild-type POLγA for primer utilization, and this will result in a heterogeneous population of short and long DNA replication products. In addition, there is a possibility that POLγA:Y955C is recruited to nicks of mtDNA and there enters an idling mode preventing ligation. Our results provide a novel explanation for the dominant mtDNA replication phenotypes seen in patients harboring the Y955C mutation, including the existence of site-specific stalling. Our data may also explain why mutations that disturb dATP pools can be especially deleterious for mtDNA synthesis.

SEA domain autoproteolysis accelerated by conformational strain: mechanistic aspects.

A subclass of SEA (sea urchin sperm protein, enterokinase, and agrin) domain proteins undergoes autoproteolysis between glycine and serine in a conserved G(-1)S+1VVV motif to generate stable heterodimers. Autoproteolysis has been suggested to involve only the intramolecular catalytic action of the conserved serine hydroxyl in combination with conformational strain of the glycine-serine peptide bond. We conducted a number of experiments and simulations on the SEA domain from the MUC1 mucin to test this mechanism. Alanine-scanning mutagenesis of polar residues in the vicinity of the cleavage site demonstrates that only the nucleophile at position +1 is required for efficient proteolysis. Molecular modeling shows that an uncleaved trans peptide is incompatible with the native heterodimeric structure, resulting in disruption of secondary structure elements and distortion of the scissile peptide bond. Insertion of glycine residues (to obtain G(n)G(-1)S+1VVV motifs) appears to relieve strain, and autoproteolysis is 100 times slower in a 1G (n=1) mutant and not measurable in 2G and 4G mutants. Removal of the catalytic serine hydroxyl hampers cleavage considerably, but measurable autoproteolysis of this S1098A mutant still proceeds in the presence of strain alone. The uncleaved SEA precursor populates interconverting partially folded conformations, and autoproteolysis coincides with adoption of proper beta-sheet secondary structure and completed folding. Molecular dynamics simulations of the precursor show that the serine hydroxyl and the preceding glycine carbonyl carbon can be in van der Waals contact at the same time as the scissile peptide bond becomes strained. These observations are all consistent with autoproteolysis accelerated by N-->O acyl shift and conformational strain imposed upon protein folding in a reaction for which the free-energy barrier is decreased by substrate destabilization rather than by transition-state stabilization. The energetics of this coupled folding and autoproteolysis mechanism is accounted for in an accompanying article.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.