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Biomedical subjects

A D Miranker

Publications and source records attributed to A D Miranker.

7 recordsLinked to original sources
PubMed2001

Kidney dialysis-associated amyloidosis: a molecular role for copper in fiber formation.

In the US alone, more than 250,000 people have impaired renal function that necessitates treatment by dialysis. A debilitating complication of long-term treatment is the deposition of beta2-microglobulin (beta2m) as amyloid fibers within the joint space. However, the intrinsic propensity of isolated beta2m protein to initiate in vitro fiber formation is negligible under conditions matched to the neutral pH and ionic conditions of serum. Here, we present evidence for a novel interaction between beta2m and Cu(2+) at a concentration within institutionally recommended limits for this metal ion in dialysate solution. Mass spectrometry, using electrospray ionization from native conditions, demonstrates that the binding of Cu(2+) is specific over Ca(2+) or Zn(2+). Despite maintaining a native-like conformation upon Cu(2+) binding, the folded protein is unusually destabilized against thermal and urea denaturation. We further demonstrate that destabilization by Cu(2+) uniquely promotes de novo fiber formation at 37 degrees C and neutral pH. Since the incidence of amyloidosis is dramatically reduced upon elimination of copper from dialysis membranes, our results provide a molecular understanding for dialysis-associated amyloid formation by beta2m.

Amyloidosis↗
PubMed2001

Islet amyloid polypeptide: identification of long-range contacts and local order on the fibrillogenesis pathway.

The pathology of type II diabetes includes deposition of amyloid in the extra cellular space surrounding the beta-cells of the endocrine pancreas. The principle component of these deposits is an insoluble fibrillar form of a normally soluble 37 residue peptide hormone, islet amyloid polypeptide. Multiple sequence analysis and peptide synthesis have identified a core set of residues (20 to 29) as intrinsically amyloidogenic. As the fibrillogenesis of the 20-29 peptide often requires conditions that deviate considerably from physiological, residues 20 to 29 may be necessary, but not sufficient, for amyloidosis. We aim to determine the structural role of residues outside this core in the context of in vitro fibrillogenesis of the wild-type peptide at physiological pH and ionic strength. Specifically, we make use of an intrinsic fluorescent probe, tyrosine 37 (Y37), to explore the role of the C terminus in fibrillogenesis. Our protocol permits steady state measurement of the lag phase and fiber conformational states of the protein under identical conditions. These are compared to a non-amyloidogenic variant of islet amyloid polypeptide from rat and N-acetyl-tyrosinamide as models of the unfolded state under matched conditions. Spectral, quenching and anisotropic properties of Y37 in the fiber state indicate that the C terminus is packed in a well-defined environment with near frozen rigidity. The presence of a fluorescence resonance energy transfer pathway shows Y37 is near F15 and F23. The lag-phase conformation, while considerably less ordered than the fiber, is more ordered than unfolded models. Differences in anisotropy between the lag and fiber state were used to monitor fibrillogenesis in real time. Parallel assessment of fiber formation using the histological dye, ThT, indicate that ordering at the C terminus of islet amyloid polypeptide is coincident with, and thus indicative of, fiber formation.

Amino Acid Sequence↗
PubMed2000

Protein complexes and analysis of their assembly by mass spectrometry.

The utility of mass spectrometry for the analysis of proteins has grown enormously in the past decade. Significant advances in detection and ionization techniques are allowing questions about noncovalent assembly to be addressed by the direct observation of gas phase complexes, their assembly in real time and their disassembly by perturbation of solution or instrument conditions. These technological innovations have plainly captured the imagination of biological researchers. Recent and novel developments include the combination of mass spectrometry with isotopic labeling, affinity labeling and genomic information. Collectively, these advances are opening new doors to the isolation of complexes, the identification of their substituents and the characterization of their conformations and assembly.

Kinetics↗
PubMed2000

Direct measurement of islet amyloid polypeptide fibrillogenesis by mass spectrometry.

A novel method for monitoring fibrillogenesis is developed and applied to the amyloidogenic peptide, islet amyloid polypeptide (IAPP). The approach, based on electrospray ionization mass spectrometry, is complementary to existing assays of fibril formation as it monitors directly the population of precursor rather than product molecules. We are able to monitor fiber formation in two modes: a quenched mode in which fibril formation is halted by dilution into denaturant and a real time mode in which fibril formation is conducted within the capillary of the electrospray source. Central to the method is the observation that fibrillar IAPP does not compromise the ionization of monomeric IAPP. Furthermore, under mild ionization conditions, fibrillar IAPP does not dissociate and contribute to the monomeric signal. Critically, we introduce an internal standard, rat IAPP, for analysis on the mass spectrometer. This standard is sufficiently similar in sequence in that it ionizes identically to human IAPP. Furthermore, the sequence is sufficiently different in that it does not form fibrils and is distinguishable on the basis of mass. Applied to IAPP fibrillogenesis, our technique reveals that precursor consumption in seeded reactions obeys first-order kinetics. Furthermore, a consistent level of monomer persists in both seeded and unseeded experiments after the fibril formation is complete. Given the inherent stability of fibrils, we expect this approach to be applicable to other amyloid systems.

Amyloid↗
PubMed1999

Global unfolding of a substrate protein by the Hsp100 chaperone ClpA.

The bacterial protein CIpA, a member of the Hsp100 chaperone family, forms hexameric rings that bind to the free ends of the double-ring serine protease ClpP. ClpA directs the ATP-dependent degradation of substrate proteins bearing specific sequences, much as the 19S ATPase 'cap' of eukaryotic proteasomes functions in the degradation of ubiquitinated proteins. In isolation, ClpA and its relative ClpX can mediate the disassembly of oligomeric proteins; another similar eukaryotic protein, Hsp104, can dissociate low-order aggregates. ClpA has been proposed to destabilize protein structure, allowing passage of proteolysis substrates through a central channel into the ClpP proteolytic cylinder. Here we test the action of ClpA on a stable monomeric protein, the green fluorescent protein GFP, onto which has been added an 11-amino-acid carboxy-terminal recognition peptide, which is responsible for recruiting truncated proteins to ClpAP for degradation. Fluorescence studies both with and without a 'trap' version of the chaperonin GroEL, which binds non-native forms of GFP, and hydrogen-exchange experiments directly demonstrate that ClpA can unfold stable, native proteins in the presence of ATP.

Adenosine Triphosphatases↗
PubMed1996

Collapse and cooperativity in protein folding.

The folding of a polypeptide chain is associated both with compactness and cooperativity within local and global regions of the protein structure, and with the formation of the native-like molecular architecture. Recent experiments shed light on these issues and their relationships to the pathways of protein folding.

Animals↗