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Andrew D Miranker

Publications and source records attributed to Andrew D Miranker.

13 recordsLinked to original sources
PubMed2006

Direct detection of transient alpha-helical states in islet amyloid polypeptide.

The protein islet amyloid polypeptide (IAPP) is a glucose metabolism associated hormone cosecreted with insulin by the beta-cells of the pancreas. In humans with type 2 diabetes, IAPP deposits as amyloid fibers. The assembly intermediates of this process are associated with beta-cell death. Here, we examine the rat IAPP sequence variant under physiological solution conditions. Rat IAPP is mechanistically informative for fibrillogenesis, as it samples intermediate-like states but does not progress to form amyloid. A central challenge was the development of a bacterial expression system to generate isotopically labeled IAPP without terminal tags, but which does include a eukaryotic post-translational modification. While optical spectroscopy shows IAPP to be natively unfolded, NMR chemical shifts of backbone and beta-carbon resonances reveal the sampling of alpha-helical states across a continuous stretch comprising approximately 40% of the protein. In addition, the manifestation of nonrandom coil chemical shifts is confirmed by the relative insensitivity of the amide proton chemical shifts to alterations in temperature. Intriguingly, the residues displaying helical propensity are conserved with the human sequence, suggesting a functional role for this conformational bias. The inability of rat IAPP to self assemble can be ascribed, in part, to several slowly exchanging conformations evident as multiple chemical shift assignments in the immediate vicinity of three proline residues residing outside of this helical region.

Amino Acid Sequence↗
PubMed2006

Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide.

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.

Amyloid↗
PubMed2006

A native to amyloidogenic transition regulated by a backbone trigger.

Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by beta-2-microglobulin (beta2m) in dialysis-related amyloidosis. Access of beta2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a beta2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamic and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type beta2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.

Amino Acid Sequence↗
PubMed2005

From chance to frequent encounters: origins of beta2-microglobulin fibrillogenesis.

It is generally accepted that amyloid formation requires partial, but not complete unfolding of a polypeptide chain. Amyloid formation by beta-2 microglobulin (beta2m), however, readily occurs under strongly native conditions provided that there is exposure to specific transition metal cations. In this review, we discuss transition metal catalyzed conformational changes in several amyloidogenic systems including prion protein, Alzheimer's and Parkinson's diseases. For some systems, including beta2m from dialysis related amyloidosis (DRA), catalysis overcomes an entropic barrier to protein aggregation. Recent data suggest that beta2m samples conformations that are under thermodynamic control, resulting in local or partial unfolding under native conditions. Furthermore, exposure to transition metal cations stabilizes these partially unfolded states and promotes the formation of small oligomers, whose structures are simultaneously near-native and amyloid-like. By serving as a tether, Cu(2+) enables the encounter of amyloidogenic conformations to occur on time scales which are significantly more rapid than would occur between freely diffusing monomeric protein. Once amyloid formation occurs, the requirement for Cu(2+) is lost. We assert that beta2m amyloid fiber formation at neutral pH may be facilitated by rearrangements catalyzed by the transient and pair wise tethering of beta2m at the blood/dialysate interface present during therapeutic hemodialysis.

Amyloid↗
PubMed2005

Quantitative measurement of fibrillogenesis by mass spectrometry.

In this chapter, a method for the quantitative determination of amyloid conversion by electrospray mass spectrometry is presented. Mass spectrometry is typically used for the purpose of measuring the mass of unknowns. However, the judicious selection of an internal standard permits the quantitative determination of protein concentration. For amyloid formation, this is particularly useful in circumstances where either the protein under study is in limited abundance, or separation of the precursor from other protein factors is impractical or undesirable. For the measurement of amyloid formation, internal standards are typically mass distinct variants of the amyloid precursor. In addition, the extreme stability of amyloid fibers permits assessment of residual precursor concentration with or without separation of fibers from unreacted precursor. Lastly, by using internal standards which are not amyloidogenic and do not interfere with fiber formation, electrospray mass spectrometry permits quantitative measurement of fiber formation in real-time and with small (pmoles) quantities of protein.

Amyloid↗
PubMed2004

Contribution of the intrinsic disulfide to the assembly mechanism of islet amyloid.

Amyloidogenesis from soluble protein requires conformational and oligomeric assembly steps. In systems where the precursor protein is natively unfolded, such as islet amyloid polypeptide (IAPP), forces and structural changes relevant to protein unfolding are not thought to participate in the assembly mechanism. Thus, fiber core structure elements should provide the dominant contributions to assembly kinetics. Here we show, however, that residues outside the amyloid core can influence the mechanism of IAPP fiber assembly. IAPP possesses an intramolecular disulfide bond between residues 2 and 7. This short-range disulfide prohibits the N-terminal region from adopting the beta-strand structure of an amyloid. We examined the role of this disulfide in fiber formation by generating a truncated construct (IAPP(8-37)) and a stable reduced form of the full-length protein (IAPP(CAM)). The fiber structures and assembly kinetics of these variants were assessed via optical and mass spectroscopy. Our data confirm that the disulfide does not contribute to the amyloid fiber core structure. Remarkably, however, it plays a central role in the assembly mechanism. First, loss of the disulfide substantially reduces fiber formation by secondary nucleation, i.e., the ability of pre-existing fibers to participate in the formation of new fibers. Second, the bypass of nucleation by seed addition is a two-step process, termed activation. Loss of the disulfide eliminates this two-step nature of seeded kinetics.

Amino Acid Sequence↗
PubMed2004

Phospholipid catalysis of diabetic amyloid assembly.

Islet amyloid polypeptide (IAPP) is a 37-residue hormone that forms cytotoxic amyloid fibers in the endocrine pancreas of patients with type II diabetes (NIDDM). A potential origin for cytotoxicity is disruption of lipid membranes by IAPP as has been observed in vitro. The cause of amyloid formation during NIDDM is not known, nor is the mechanism by which membrane disruption occurs in vitro. Here, we use kinetic studies in conjunction with assessments of lipid binding and electron microscopy to investigate the interactions of IAPP with phospholipid bilayers and the morphological effects of membranes on IAPP fibers. Fibrillogenesis of IAPP is catalyzed by synthetic and human tissue-derived phospholipids, leading to >tenfold increases in the rate of fibrillogenesis. The molecular basis of this phenomenon includes a strong dependence on the concentration and charge density of the membrane. IAPP binds to lipid membranes of mixed anionic (DOPG) and zwitterionic (DOPC) content. The transition for binding occurs over a physiologically relevant range of anionic content. Membrane binding by IAPP occurs on timescales that are short compared to fibrillogenesis and results in assembly into preamyloid states via ordered interactions at the N but not C terminus of the protein. These assemblies lead both to gross morphological changes in liposomes and to alterations in the appearance of early fibers when compared to liposome-free fibril formation. Intact bilayer surfaces are regenerated upon dissociation of fibers from the membrane surface. These findings offer a structural mechanism of membrane destabilization and suggest that changes in lipid metabolism could induce IAPP fiber formation in NIDDM.

Amino Acid Sequence↗
PubMed2004

Oligomeric assembly of native-like precursors precedes amyloid formation by beta-2 microglobulin.

The deposition of beta-2-microglobulin (beta2m) as amyloid fibers results in debilitating complications for renal failure patients who are treated by hemodialysis. In vitro, wild-type beta2m can be converted to amyloid under physiological conditions by exposure to biomedically relevant concentrations of Cu(2+). In this work, we have made comparative measurements of the structural and oligomeric changes in beta2m at time points preceding fibrillogenesis. Our results show Cu(2+) mediates the formation of a monomeric, activated state followed by the formation of a discrete dimeric intermediate. The dimeric intermediates then assemble into tetra- and hexameric forms which display little additional oligomerization on the time scales of their own formation (<1 h). Amyloid fiber formation progresses from these intermediate states but on much longer time scales (>1 week). Although Cu(2+) is necessary for the generation and stabilization of these intermediates, it is not required for the stability of mature amyloid fibers. This suggests that Cu(2+) acts as an initiating factor of amyloidosis by inducing oligomer formation. (1)H NMR and near-UV circular dichroism are used to establish that oligomeric intermediates are native-like in structure. The native-like structure and discrete oligomeric size of beta2m amyloid intermediates suggest that this protein forms fibrils by structural domain swapping.

Amyloid↗
PubMed2004

The mechanism of insulin action on islet amyloid polypeptide fiber formation.

The pathology of type II diabetes includes the presence of cytotoxic amyloid deposits in the islets of Langerhans. The main component of these deposits, islet amyloid polypeptide (IAPP), is a hormone involved in glucose metabolism and is normally co-secreted with insulin by the beta-cells of the pancreas. Here, we perform in vitro IAPP fibrillogenesis experiments in the presence and in the absence of insulin to elucidate the mechanism by which insulin acts on fiber formation. We find that insulin is an exceptionally potent inhibitor. In contrast to the vast excess of insulin over IAPP in vivo, substoichiometric amounts of insulin inhibit seeded and unseeded reactions by more than tenfold in vitro. Unusually, the magnitude of the inhibitory effect is dependent on the concentration of insulin, yet independent of the concentration of IAPP. In addition, insulin appears to bind non-specifically to fiber surfaces, giving rise to altered morphology. IAPP fiber formation in vitro requires a minimum of three steps: fiber-independent nucleation, elongation, and fiber-dependent nucleation. Furthermore, these steps are attenuated by the presence of a dispersed-phase transition. We interpret these data in the context of the phase-mediated fibrillogenesis model (PMF) and conclude through experiment and kinetic simulation that the dominant effect of insulin is to act on the elongation portion of the reaction. These results suggest that amyloid formation in type II diabetes involves either an additional agent that acts as an accelerant, or a step that segregates IAPP from insulin.

Amyloid↗
PubMed2002

Formation of a copper specific binding site in non-native states of beta-2-microglobulin.

A debilitating complication of long-term hemodialysis is the deposition of beta-2-microglobulin (beta2m) as amyloid plaques in the joint space. We have recently shown that Cu(2+) can be a contributing, if not causal, factor at concentrations encountered during dialysis therapy. The basis for this effect is destabilization and incorporation of beta2m into amyloid fibers upon binding of Cu(2+). In this work, we demonstrate that while beta2m binds Cu(2+) specifically in the native state, it is binding of Cu(2+) by non-native states of beta2m which is responsible for destabilization. Mutagenesis of potential coordinating groups for Cu(2+) shows that native state binding of Cu(2+) is mediated by residues and structures that are different than those which bind in non-native states. An increased affinity for copper by non-native states compared to that of the native state gives rise to overall destabilization. Using mass spectrometry, NMR, and fluorescence techniques, we show that native state binding is localized to H31 and W60 and is highly specific for Cu(2+) over Zn(2+) and Ni(2+). Binding of Cu(2+) in non-native states of beta2m is mediated by residues H13, H51, and H84, but not H31. Although denatured beta2m has characteristics of a globally unfolded state, it nevertheless demonstrates the following strong specificity of binding: Cu(2+) > Zn(2+) >> Ni(2+). This requires the existence of a well-defined structure in the unfolded state of this protein. As Cu(2+) effects are reported in many other amyloidoses, e.g., PrP, alpha-synuclein, and Abeta, our results may be extended to the emerging field of divalent ion-associated amyloidosis.

Amino Acid Sequence↗
PubMed2002

Islet amyloid: phase partitioning and secondary nucleation are central to the mechanism of fibrillogenesis.

Islet amyloid polypeptide (IAPP) contributes to the pathogenesis of type II diabetes by depositing as cytotoxic amyloid fibers in the endocrine pancreas. Fiber formation occurs with a marked conformational change from an unstructured precursor. Using real-time quantitative kinetic methods, fibrillogenesis was characterized as a function of protein, denaturant, and seed concentration. Several observations are in sharp contrast to the expectations for nucleation-dependent polymerization. First, the half-time of conversion for both de novo and seeded kinetics were found to be independent of protein concentration. Second, while elongation kinetics scale linearly with protein concentration, they are relatively insensitive to changes in the total seed concentration. Third, seeded bypass of de novo fiber formation kinetics shows a lag phase. The seeded lag phase is eliminated by a time delay before the introduction of seed to a de novo reaction. Last, conversion is highly cooperative, with the time required for 10-90% conversion occurring much faster than the lag time. At a minimum, four kinetic steps are required to describe these observations: activation, fiber independent nucleation, fiber-dependent nucleation, and elongation. Furthermore, we invoke a phase transition in which protein initially forms an off-pathway dispersion. This single construct allows us to model both the concentration independence of the de novo reaction time and the first-order concentration dependence of the elongation kinetics. Marked acceleration of this reaction by hexafluoro-2-propanol reinforces this view by altering the relative solubility of the two phases and/or by stabilizing hydrogen-bonded structures in the transition states of the reaction pathway.

Amyloid↗