The Significance of the Location of Mutations for the Native-State Dynamics of Human Lysozyme

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.This research was supported by the Biotechnology and Biological Sciences Research Council (BB/E019927/1 to C.M.D., C.V.R., and J.R.K.), the Medical Research Council (E.D.G. and C.M.D.), the Belgian Program of Interuniversity Attraction Poles administered by the Federal Office for Scientific Technical and Cultural Affairs (PAI numbers P6/19 and P7144 to C.M.D. and M.D.), the European Union’s Sixth Framework Program (LSHM-CT-2006-037525 to C.M.D. and M.D.), and Programme grants from the Wellcome Trust and the Leverhulme Trust (C.M.D.). It was also supported by a Korean Government Scholarship for Overseas Studies (M.A.), the Winston Churchill Foundation (C.L.H.), and Boerhinger Ingleheim funds (A.D.). The NMR facility at the Department of Chemistry, University of Cambridge, is supported in part by an EPSRC Core Capability grant (EP/K039520/1)

Application of Lysine-specific Labeling to Detect Transient Interactions Present During Human Lysozyme Amyloid Fibril Formation.

Populating transient and partially unfolded species is a crucial step in the formation and accumulation of amyloid fibrils formed from pathogenic variants of human lysozyme linked with a rare but fatal hereditary systemic amyloidosis. The partially unfolded species possess an unstructured β-domain and C-helix with the rest of the α-domain remaining native-like. Here we use paramagnetic relaxation enhancement (PRE) measured by NMR spectroscopy to study the transient intermolecular interactions between such intermediate species. Nitroxide spin labels, introduced specifically at three individual lysine residues, generate distinct PRE profiles, indicating the presence of intermolecular interactions between residues within the unfolded β-domain. This study describes the applicability to PRE NMR measurements of selective lysine labeling, at different sites within a protein, as an alternative to the introduction of spin labels via engineered cysteine residues. These results reveal the importance of the β-sheet region of lysozyme for initiating self-assembly into amyloid fibrils

Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aβ) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aβ or small soluble Aβ oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (α2M), although the chaperone activity of α2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aβ, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding.Wellcome Trust Programme Grant 094425/Z/10/

The significance of the location of mutations for the native-state dynamics of human lysozyme

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation

Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer'sassociated amyloid beta peptide

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aß) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aß or small soluble Aß oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (a2M), although the chaperone activity of a2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aß, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding

Molecular Structure, Biosynthesis, and Distribution of Coenzyme Q

Coenzyme Q is a very old molecule in evolutionary terms that has accumulated numerous functions in the cellular metabolism beyond its primordial function, the electron transport. In all organisms, coenzyme Q maintains a highly conserved structure allowing a localization inside cell membranes in a hydrophobic environment thanks to having an isoprenoid tail, and at the same time allows the polar ring benzene to interact with acceptors and electron donors. Coenzyme Q deficiency constitutes a group of mitochondrial diseases. Affected patients suffer mainly a decrease in energy production that induces dysfunctions in most organs and body systems. Current therapeutic alternatives are based on increasing coenzyme Q levels either through induction of endogenous mechanisms or exogenous supplementation. This chapter includes both aspects, the mechanisms associated with the coenzyme Q supplementation and the regulatory mechanisms of coenzyme Q biosynthesis. In terms of synthesis, the structure of coenzyme Q is complicated since it requires the participation of two well-differentiated pathways that must be carefully regulated. The synthesis is carried out through the participation of a multienzyme complex located in the inner mitochondrial membrane and controlled by different levels of regulation that at this time are not well-known