Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers.

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several µmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.

A kidney resident macrophage subset is a candidate biomarker for renal cystic disease in preclinical models.

Although renal macrophages have been shown to contribute to cyst development in polycystic kidney disease (PKD) animal models, it remains unclear whether there is a specific macrophage subpopulation involved. Here, we analyzed changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We observed that CD206+ resident macrophages were minimal in a normal adult kidney but accumulated in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and showed that this significantly reduced cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increased in kidneys and in the urine from autosomal-dominant PKD (ADPKD) patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation, and reveal that the CD206+ resident macrophages in urine could serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients. This article has an associated First Person interview with the first author of the paper.

Apolipoprotein E4 exhibits intermediates with domain interaction.

ApoE4(C112R) is the strongest risk factor for Alzheimer's disease, while apoE3(C112) is considered normal. The C112R substitution is believed to alter the interactions between the N-terminal (NTD) and the C-terminal domain (CTD) leading to major functional differences. Here we investigate how the molecular property of the residue at position 112 affects domain interaction using an array of C112X substitutions with arginine, alanine, threonine, valine, leucine and isoleucine as 'X'. We attempt to determine the free energy of domain interaction (∆GINT) from stabilities of the NTD (∆GNTD) and CTD (∆GCTD) in the full-length apoE, and the stabilities of fragments of the NTD (∆GNTF) and CTD (∆GCTF), using the relationship, ∆GINT = ∆GNTD + ∆GCTD - ∆GNTF - ∆GCTF. We find that although ∆GNTD is strongly dependent on the C112X substitutions, ∆GNTD - ∆GNTF is small. Furthermore, ∆GCTD remains nearly the same as ∆GCTF. Therefore, ∆GINT is estimated to be small and similar for the apoE isoforms. However, stability of domain interaction monitored by urea dependent changes in interdomain Forster Resonance Energy Transfer (FRET) is found to be strongly dependent on C112X substitutions. ApoE4 exhibits the highest mid-point of denaturation of interdomain FRET. To resolve the apparently contradictory observations, we hypothesize that higher interdomain FRET in apoE4 in urea may involve 'intermediate' states. Enhanced fluorescence of bis-ANS and susceptibility to proteolytic cleavage support that apoE4, specifically, the NTD of apoE4 harbor 'intermediates' in both native and mildly denaturing conditions. The intermediates could hold key to the pathological functions of apoE4.