studies-chaperone-cochaperone-interactions-using-homogenous-bead

Studies of Chaperone-Cochaperone Interactions using Homogenous Bead-Based Assay

Targeting the heat shock protein 90 (Hsp90)-cochaperone interactions provides the possibility to specifically regulate Hsp90-dependent intracellular ...

Karolinska Institutet

The amyloid beta-peptide (Abeta) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Abeta and accumulation of Abeta has been detected in mitochondria. Because Abeta is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Abeta uptake. Our results from rat mitochondria show that Abeta is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Abeta association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Abeta to mitochondrial cristae. A similar distribution pattern of Abeta in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Abeta in mitochondria and demonstrate both in vitro and in vivo that Abeta is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Abeta can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Abeta, a peptide considered to be of major significance in Alzheimer's disease.

The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae.

Accumulating evidence suggest that alterations in energy metabolism are among the earliest events that occur in the Alzheimer disease (AD) affected brain. Energy consumption is drastically decreased in the AD-affected regions of cerebral cortex and hippocampus pointing towards compromised mitochondrial function of neurons within specific brain regions. This is accompanied by an elevated production of reactive oxygen species contributing to increased rates of neuronal loss in the AD-affected brain regions. In this review, we will discuss the role of mitochondrial function and dysfunction in AD. We will focus on the consequences of amyloid precursor protein and amyloid-beta peptide accumulation in mitochondria and their involvement in AD pathogenesis.

Mitochondrial accumulation of APP and Abeta: significance for Alzheimer disease pathogenesis.

Omi/HtrA2, a mitochondrial serine protease with chaperone activity, is involved in varied intracellular processes. Dysfunctional Omi/HtrA2 has thus been implicated in various neurodegenerative disorders. Previously, we have shown that γ-secretase complexes are present and active in mitochondria. Here, we demonstrate that peptide corresponding to C-terminus of presenilin-1, as previously reported to activate Omi/HtrA2, interacts with Omi/HtrA2 in isolated mitochondria. Moreover, we show that Omi/HtrA2 interacts with presenilin in active γ-secretase complexes located to mitochondria. Using a biotinylated γ-secretase inhibitor and confocal microscopy, we could further confirm the association of γ-secretase complexes with mitochondrial Omi/HtrA2. Furthermore, determination of γ-secretase complex topology in isolated mitochondria revealed an association of γ-secretase complexes with the outer membrane of mitochondria with the extreme PS1 C-terminus facing the inter-membrane space. We have also studied the impact of Omi/HtrA2 on γ-secretase activity, measuring APP intracellular domain (AICD) production. We found reduced AICD production in mitochondria isolated from Omi/HtrA2 knockout mouse embryonic fibroblasts, indicating a significant role of Omi/HtrA2 on γ-secretase activity. Thus, our results provide information for understanding the interplay between mitochondrial Omi/HtrA2 and γ-secretase complexes in AD.

Association of Omi/HtrA2 with γ-secretase in mitochondria.

Intracellular amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.

Mitochondrial γ-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein.

Previous findings demonstrated an altered pattern of amyloid-β protein precursor (AβPP) expression in platelets of Alzheimer's disease (AD) patients compared with either healthy control subjects or patients with non-Alzheimer-type dementia. In an attempt to explore the diagnostic potential of platelet AβPP metabolism, we have generated monoclonal antibodies directed to the N-terminal part of AβPP. We have observed two different antibody recognition patterns of AβPP: one resembling previously described 130 kDa and 105 kDa species and a novel AβPP 115 kDa form. This form was significantly increased in platelets of the mild cognitive impairment and AD group as compared to control subjects. The abundance of AβPP 115 kDa species correlated with the previously described AβPP 130/105 kDa ratio as well as with Mini-Mental State Examination score. Despite the inability of these particular monoclonal antibodies to recognize native forms of AβPP, identification of a new AβPP isoform in platelets as a potential AD biomarker can provide an additional tool for the development of a reliable diagnostic test to detect preclinical stages of AD.

Abnormal platelet amyloid-β protein precursor (AβPP) metabolism in Alzheimer's disease: identification and characterization of a new AβPP isoform as potential biomarker.

Accumulation of amyloid β-peptide (Aβ) in the brain is an important event in the pathogenesis of Alzheimer disease. We have used a transgenic mouse model expressing human amyloid precursor protein (APP) with the Arctic mutation to investigate whether Aβ deposition is correlated with mitochondrial functions in these animals. We found evidence of mitochondrial dysfunction (i.e., decreased mitochondrial membrane potential, increased production of reactive oxygen species and oxidative DNA damage) at 6 months of age, when the mice showed very mild Aβ deposition. More pronounced mitochondrial abnormalities were present in 24-month-old TgAPParc mice with more extensive Aβ pathology. This study demonstrates for the first time mitochondrial dysfunction in transgenic mice with a mutation within the Aβ peptide (the Arctic APP mutation), and confirms previous studies suggesting that mitochondrial dysfunction and oxidative stress is an early event in the pathogenesis of Alzheimer disease. This study demonstrates mitochondrial dysfunction in transgenic mice with a mutation within the amyloid beta (Aβ) peptide (the Arctic amyloid precursor protein (APP) mutation). We found evidence of mitochondrial dysfunction (i.e. decreased mitochondrial membrane potential (MMP), increased production of reactive oxygen species (ROS) and oxidative DNA damage) at 6 months of age, when very mild Aβ deposition is present in the mice. Also, the cytochrome c (COX) activity was significantly decreased in mitochondria from transgenic mice at 24 months of age.

Mitochondrial dysfunction in a transgenic mouse model expressing human amyloid precursor protein (APP) with the Arctic mutation.

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer's disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.

Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51.

Mitochondrial dysfunction is an early feature of Alzheimer's disease (AD) and may play an important role in the pathogenesis of disease. It has been shown that amyloid beta peptide (Aβ) and amyloid precursor protein (APP) interact with mitochondria contributing to the mitochondrial dysfunction in AD. Prevention of abnormal protein targeting to mitochondria can protect normal mitochondrial function, increase neuronal survival and at the end, ameliorate symptoms of AD and other neurodegenerative disorders. First steps of mitochondrial protein import are coordinated by molecular chaperones Hsp70 and Hsp90 that bind to the newly synthesized mitochondria-destined proteins and deliver them to the protein import receptors on the surface of organelle. Here, we have described the development of a novel compound named GMP-1 that disrupts interactions between Hsp70/Hsp90 molecular chaperones and protein import receptor Tom70. GMP-1 treatment of SH-SY5Y cells results in decrease in mitochondria-associated APP and protects SH-SY5Y cells from toxic effect of Aβ exposure. Experiments in drosophila and mice models of AD demonstrated neuroprotective effect of GMP-1 treatment, improvement in memory and behaviour tests as well as restoration of mitochondrial function.

Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease.

Dicarboxylate clamp tetratricopeptide repeat (dcTPR) motif-containing proteins are well-known partners of the heat shock protein (Hsp) 70 and Hsp90 molecular chaperones. Together, they facilitate a variety of intracellular processes, including protein folding and maturation, protein targeting, and protein degradation. An extreme C-terminal sequence, the EEVD motif, is identical in Hsp70 and Hsp90, and is indispensable for their interaction with dcTPR proteins. However, almost no information is available on the existence of other potential dcTPR-interacting proteins. We searched the human protein database for proteins with C-terminal sequences similar to that of Hsp70/Hsp90 to identify potential partners of dcTPR proteins. The search identified 112 proteins containing a Hsp70/Hsp90-like signature at their C termini. Gene Ontology enrichment analysis of identified proteins revealed enrichment of distinct protein classes, such as molecular chaperones and proteins of the ubiquitin-proteasome system, highlighting the possibility of functional specialization of proteins containing a Hsp70/Hsp90-like signature. We confirmed interactions of selected proteins containing Hsp70/Hsp90-like C termini with dcTPR proteins both  and . Analysis of interactions of 10-amino-acid peptides corresponding to the C termini of identified proteins with dcTPR proteins revealed significant differences in binding strength between various peptides. We propose a hierarchical mode of interaction within the dcTPR protein network. These findings describe a novel dcTPR protein interaction networks and provide a rationale for selective regulation of protein-protein interactions within this network.

 identification and biochemical characterization of the human dicarboxylate clamp TPR protein interaction network.

Alzheimer's disease (AD) represents a major public health threat and, unfortunately, available therapeutics provide only temporary symptomatic relief. AD is a complex multifactorial disease and failure of single target therapeutics targeting amyloid-β (Aβ) in recent clinical trials suggests that future AD drug development should be focused on simultaneous targeting of several pathological hallmarks of the disease. Recently, we have shown that GMP-1, a 2-(methoxymethyl)pyrimido [1, 2-a] benzimidazol-4-ol, protects mitochondrial function in drosophila and mice models of AD, and improved memory and behavior indicating neuroprotective effect of GMP-1 treatment. Here, we have found that GMP-1 specifically binds to copper and zinc, metals that are dysregulated in AD brain. Addition of GMP-1 does not inhibit metal-dependent enzymatic reactions. Also, binding of Zn(II) and Cu(II) by GMP-1 is weaker than the 8-hydroxyquinoline scaffold compound clioquinol previously tested in AD clinical trials. However, GMP-1 affects Cu(II)-dependent Aβ fibrillization as well as oxidative damage and viability of SH-SY5Y cells upon addition of Cu(II) and Aβ. Our data provide new insight on GMP-1 as a Zn(II) and Cu(II) specific metal chelator of moderate affinity that can be responsible for some of its neuroprotective effects observed in AD animal models.

Metal Binding by GMP-1 and Its Pyrimido [1, 2]benzimidazole Analogs Confirms Protection Against Amyloid-β Associated Neurotoxicity.

Alzheimer's disease (AD) is the most common form of dementia characterized by presence of extracellular amyloid plaques and intracellular neurofibrillary tangles composed of tau protein. Currently there are close to 50 million people living with dementia and this figure is expected to increase to 75 million by 2030 putting a huge burden on the economy due to the health care cost. Considering the effects on quality of life of patients and the increasing burden on the economy, there is an enormous need of new disease modifying therapies to tackle this disease. The current therapies are dominated by only symptomatic treatments including cholinesterase inhibitors and N-methyl-D-aspartate receptor blockers but no disease modifying treatments exist so far. After several failed attempts to develop drugs against amyloidopathy, tau targeting approaches have been in the main focus of drug development against AD. After an overview of the tauopathy in AD, this review summarizes recent findings on the development of small molecules as therapeutics targeting tau modification, aggregation, and degradation, and tau-oriented multi-target directed ligands. Overall, this work aims to provide a comprehensive and critical overview of small molecules which are being explored as a lead candidate for discovering drugs against tauopathy in AD.

Article	Year
The amyloid beta-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18757748	2008
Mitochondrial accumulation of APP and Abeta: significance for Alzheimer disease pathogenesis. Journal of cellular and molecular medicine\| PubMed ID: 19725915	2009
Association of Omi/HtrA2 with γ-secretase in mitochondria. Neurochemistry international\| PubMed ID: 20705111	2010
Mitochondrial γ-secretase participates in the metabolism of mitochondria-associated amyloid precursor protein. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 20833873	2011
Abnormal platelet amyloid-β protein precursor (AβPP) metabolism in Alzheimer's disease: identification and characterization of a new AβPP isoform as potential biomarker. Journal of Alzheimer's disease : JAD\| PubMed ID: 23388171	2013
Mitochondrial dysfunction in a transgenic mouse model expressing human amyloid precursor protein (APP) with the Arctic mutation. Journal of neurochemistry\| PubMed ID: 26500157	2016
Combined x-ray crystallography and computational modeling approach to investigate the Hsp90 C-terminal peptide binding to FKBP51. Scientific reports\| PubMed ID: 29079741	2017
Development of GMP-1 a molecular chaperone network modulator protecting mitochondrial function and its assessment in fly and mice models of Alzheimer's disease. Journal of cellular and molecular medicine\| PubMed ID: 29704317	2018
identification and biochemical characterization of the human dicarboxylate clamp TPR protein interaction network. FEBS open bio\| PubMed ID: 30410862	2018
Metal Binding by GMP-1 and Its Pyrimido [1, 2]benzimidazole Analogs Confirms Protection Against Amyloid-β Associated Neurotoxicity. Journal of Alzheimer's disease : JAD\| PubMed ID: 31839606	2020
Small molecule therapeutics for tauopathy in Alzheimer's disease: Walking on the path of most resistance. European journal of medicinal chemistry\| PubMed ID: 33139110	2021

Pavel F. Pavlov

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