5 results on '"Sreelakshmi Cherakara"'
Search Results
2. CD206+ resident macrophages are a candidate biomarker for renal cystic disease in preclinical models and patients with ADPKD
- Author
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Zhang, Li, Kurt A, Zimmerman, Sreelakshmi, Cherakara, Phillip, Chumley, James F, Collawn, Jun, Wang, Courtney J, Haycraft, Cheng J, Song, Teresa, Chacana, Reagan S, Andersen, Mandy J, Croyle, Ernald J, Aloria, Raksha P, Hombal, Isis N, Thomas, Hanan, Chweih, Kristin L, Simanyi, James F, George, John M, Parant, Michal, Mrug, and Bradley K, Yoder
- Abstract
Although renal macrophages have been shown to contribute to cystic kidney disease in PKD animal models, it remains unclear if there is a specific macrophage subpopulation involved. Here we analyze changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We demonstrate that CD206+ resident macrophages are minimal in a normal adult kidney but accumulate in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and show this significantly reduces cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increases in kidneys and in the urine from ADPKD patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation and demonstrate that the CD206+ resident macrophages in urine may serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients.
- Published
- 2022
3. Hsp70 Inhibits Aggregation of IAPP by Binding to the Heterogeneous Prenucleation Oligomers
- Author
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Mithun Maddheshiya, Bankanidhi Sahoo, Sreelakshmi Cherakara, Timir Baran Sil, Kanchan Garai, S. Deepa, and Neeraja Chilukoti
- Subjects
Amyloid ,endocrine system ,0303 health sciences ,Chemistry ,Size-exclusion chromatography ,Biophysics ,Fluorescence correlation spectroscopy ,Articles ,Intrinsically disordered proteins ,Oligomer ,Islet Amyloid Polypeptide ,03 medical and health sciences ,chemistry.chemical_compound ,Amyloid disease ,0302 clinical medicine ,Förster resonance energy transfer ,Monomer ,HSP70 Heat-Shock Proteins ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
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.
- Published
- 2021
4. Hsp70 inhibits aggregation of Islet amyloid polypeptide by binding to the heterogeneous prenucleation oligomers
- Author
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Mithun Maddheshiya, Neeraja Chilukoti, Sreelakshmi Cherakara, Kanchan Garai, S. Deepa, and Bankanidhi Sahoo
- Subjects
geography ,endocrine system ,geography.geographical_feature_category ,Amyloid ,Size-exclusion chromatography ,Multiangle light scattering ,Fluorescence correlation spectroscopy ,Islet ,Intrinsically disordered proteins ,chemistry.chemical_compound ,Amyloid disease ,Monomer ,chemistry ,Biophysics - Abstract
Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, mechanism of the interactions of Hsp70 with the amyloidogenic intrinsically disordered proteins (IDPs) is not clear. Here, we use Hsp70 from different organisms to show that it inhibits aggregation of Islet amyloid polypeptide (IAPP) at substoichiometric concentrations even in absence of ATP. The effect is found to be the strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating role of Hsp70 in preventing primary nucleation possiblyviainteractions with the prefibrillar oligomers of IAPP. Fluorescence Correlation Spectroscopy (FCS) measurements of the solutions containing fluorescently labelled Hsp70 and IAPP exhibit fluorescence bursts suggesting formation of heterogeneous complexes of oligomeric IAPP binding to multiple molecules of Hsp70. Size exclusion chromatography and field flow fractionation are then used to fractionate the smaller complexes. Multiangle light scattering and FCS measurements suggest that these complexes comprise of monomers of Hsp70 and small oligomers of IAPP. However, concentration of the complexes is measured to be a few nanomolar amidst several μmolar of free Hsp70 and IAPP. Hence, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a common feature of the interactions between the chaperones and the amyloidogenic IDPs. While strong interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers avert interference with the functions of the IDPs.
- Published
- 2020
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5. Apolipoprotein E4 exhibits intermediates with domain interaction
- Author
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Sreelakshmi Cherakara, Kanchan Garai, and Subhrajyoti Dolai
- Subjects
Models, Molecular ,Apolipoprotein E4 ,Apolipoprotein E3 ,Biophysics ,Biochemistry ,Analytical Chemistry ,Structure-Activity Relationship ,03 medical and health sciences ,Valine ,Humans ,Protein Isoforms ,Protein Interaction Domains and Motifs ,Threonine ,Molecular Biology ,Protein Unfolding ,030304 developmental biology ,Alanine ,0303 health sciences ,Protein Stability ,Chemistry ,030302 biochemistry & molecular biology ,Molten globule ,Kinetics ,Förster resonance energy transfer ,Mutation ,CTD ,Leucine ,Isoleucine ,Protein Binding - Abstract
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.
- Published
- 2020
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