Atta Ahmad

Early Events in the Fibrillation of Monomeric Insulin

Insulin has a largely α-helical structure and exists as a mixture of hexameric, dimeric, and monomeric states in solution, depending on the conditions: the protein is monomeric in 20% acetic acid. Insulin forms amyloid-like fibrils under a variety of conditions, especially at low pH. In this study we investigated the fibrillation of monomeric human insulin by monitoring changes in CD, attenuated total reflectance-Fourier transform infrared spectroscopy, 8-anilinonaphthalenesulfonic acid fluorescence, thioflavin T fluorescence, dynamic light scattering, and H/D exchange during the initial stages of the fibrillation process to provide insight into early events involving the monomer. The results demonstrate the existence of structural changes occurring before the onset of fibril formation, which are detectable by multiple probes. The data indicate at least two major populations of oligomeric intermediates between the native monomer and fibrils. Both have significantly non-native conformations, and indicate that fibrillation occurs from a betarich structure significantly distinct from the native fold.

Guiding Protein Aggregation with Macromolecular Crowding

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.

Allostery in the Hsp70 chaperone proteins.

Heat shock 70-kDa (Hsp70) chaperones are essential to in vivo protein folding, protein transport, and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy, and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimers and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed.

Heat shock protein 70 kDa chaperone/DnaJ cochaperone complex employs an unusual dynamic interface

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70–DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1–70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual “tethered” binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein. Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422–433].

Inhibitors of Difficult Protein–Protein Interactions Identified by High-Throughput Screening of Multiprotein Complexes

Protein-protein interactions (PPIs) are important in all aspects of cellular function, and there is interest in finding inhibitors of these contacts. However, PPIs with weak affinities and/or large interfaces have traditionally been more resistant to the discovery of inhibitors, partly because it is more challenging to develop high-throughput screening (HTS) methods that permit direct measurements of these physical interactions. Here, we explored whether the functional consequences of a weak PPI might be used as a surrogate for binding. As a model, we used the bacterial ATPase DnaK and its partners DnaJ and GrpE. Both DnaJ and GrpE bind DnaK and catalytically accelerate its ATP cycling, so we used stimulated nucleotide turnover to indirectly report on these PPIs. In pilot screens, we identified compounds that block activation of DnaK by either DnaJ or GrpE. Interestingly, at least one of these molecules blocked binding of DnaK to DnaJ, while another compound disrupted allostery between DnaK and GrpE without altering the physical interaction. These findings suggest that the activity of a reconstituted multiprotein complex might be used in some cases to identify allosteric inhibitors of challenging PPIs.

The Mechanism of Enhanced Insulin Amyloid Fibril Formation by NaCl Is Better Explained by a Conformational Change Model

The high propensity of insulin to fibrillate causes severe biomedical and biotechnological complications. Insulin fibrillation studies attain significant importance considering the prevalence of diabetes and the requirement of functional insulin in each dose. Although studied since the early years of the 20th century, elucidation of the mechanism of insulin fibrillation has not been understood completely. We have previously, through several studies, shown that insulin hexamer dissociates into monomer that undergoes partial unfolding before converting into mature fibrils. In this study we have established that NaCl enhances insulin fibrillation mainly due to subtle structural changes and is not a mere salt effect. We have carried out studies both in the presence and absence of urea and Gdn.HCl and compared the relationship between conformation of insulin induced by urea and Gdn.HCl with respect to NaCl at both pH 7.4 (hexamer) and pH 2 (monomer). Fibril formation was followed with a Thioflavin T assay and structural changes were monitored by circular dichroism and size-exclusion chromatography. The results show salt-insulin interactions are difficult to classify as commonly accepted Debye-Hückel or Hofmeister series interactions but instead a strong correlation between the association states and conformational states of insulin and their propensity to fibrillate is evident.

Reply to Sousa et al.: Evaluation of competing J domain:Hsp70 complex models in light of methods used

We respond to Sousa et al. (1) who compare a crystal structure (2) of a disulfide-linked adduct of human Hsp70 nucleotide binding domain (NBD) and auxilin J domain, with the solution complex (3) of Escherichia coli Hsp70 NBD, substrate binding domain (SBD), substrate and DnaJ J domain. The molecular models are very different despite >60% sequence homology (the coordinates in ref. 3 are available on request).

The disorderly conduct of Hsc70 and its interaction with the Alzheimer's-related Tau protein

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71-kDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein Tau, which is linked to Alzheimers disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70s chaperone activity or allostery in vitro. Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the Tau sequence GKVQIINKKG (with a KD = 500 nm) causes dramatic rigidification of BETA. NOE distance measurements revealed that Tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second Tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nm), whereas the others do not (KD > 100 μm). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides.