Showing papers by "Jason E. Gestwicki published in 2012"

Guidelines for the use and interpretation of assays for monitoring autophagy

Abstract: In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo

Abstract: More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy....

Features of protein-protein interactions that translate into potent inhibitors: topology, surface area and affinity

Abstract: Protein-protein interactions (PPIs) control the assembly of multi-protein complexes and, thus, these contacts have enormous potential as drug targets. However, the field has produced a mix of both exciting success stories and frustrating challenges. Here, we review known examples and explore how the physical features of a PPI, such as its affinity, hotspots, off-rates, buried surface area and topology, might influence the chances of success in finding inhibitors. This analysis suggests that concise, tight binding PPIs are most amenable to inhibition. However, it is also clear that emerging technical methods are expanding the repertoire of 'druggable' protein contacts and increasing the odds against difficult targets. In particular, natural product-like compound libraries, high throughput screens specifically designed for PPIs and approaches that favour discovery of allosteric inhibitors appear to be attractive routes. The first group of PPI inhibitors has entered clinical trials, further motivating the need to understand the challenges and opportunities in pursuing these types of targets.

Allostery in the Hsp70 chaperone proteins.

Abstract: 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 Alzheimer’s and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed.

Hsp70 Protein Complexes as Drug Targets

Abstract: Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70's interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, including roles in pro-folding, pro-degradation and pro-trafficking pathways. Thus, a promising strategy may be to block protein- protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to these goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.

Fine-tuning multiprotein complexes using small molecules.

Abstract: Multiprotein complexes such as the transcriptional machinery, signaling hubs, and protein folding machines are typically composed of at least one enzyme combined with multiple non-enzymes. Often the components of these complexes are incorporated in a combinatorial manner, in which the ultimate composition of the system helps dictate the type, location, or duration of cellular activities. Although drugs and chemical probes have traditionally targeted the enzyme components, emerging strategies call for controlling the function of protein complexes by modulation of protein–protein interactions (PPIs). However, the challenges of targeting PPIs have been well documented, and the diversity of PPIs makes a “one-size-fits-all” solution highly unlikely. These hurdles are particularly daunting for PPIs that encompass large buried surface areas and those with weak affinities. In this Review, we discuss lessons from natural systems, in which allostery and other mechanisms are used to overcome the challenge of regulat...

Protein quality control in neurodegenerative disease.

Abstract: The accumulation of misfolded proteins is a common feature of many neurodegenerative diseases. These observations suggest a potential link between these disorders and protein quality control, a collection of cellular pathways that sense damage to proteins and facilitate their turnover. Consistent with this idea, activation of quality control components, such as molecular chaperones, has been shown to be protective in multiple neurodegenerative disease models. In addition, key studies have suggested that quality control deteriorates with age, further supporting a relationship between these processes. In this chapter, we discuss the evidence linking neurodegeneration to quality control and present the emerging models. We also speculate on why proper quality control is so difficult for certain proteins.

Analysis of the Tau-Associated Proteome Reveals That Exchange of Hsp70 for Hsp90 Is Involved in Tau Degradation

Abstract: The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in 15 neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 min after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the data set was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer's disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis.

Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK)

Abstract: The molecular chaperone DnaK binds to exposed hydrophobic segments in proteins, protecting them from aggregation. DnaK interacts with protein substrates via its substrate-binding domain, and the affinity of this interaction is allosterically regulated by its nucleotide-binding domain. In addition to regulating interdomain allostery, the nucleotide state has been found to influence homo-oligomerization of DnaK. However, the architecture of oligomeric DnaK and its potential functional relevance in the chaperone cycle remain undefined. Towards that goal, we examined the structures of DnaK by negative stain electron microscopy. We found that DnaK samples contain an ensemble of monomers, dimers, and other small, defined multimers. To better understand the function of these oligomers, we stabilized them by cross-linking and found that they retained ATPase activity and protected a model substrate from denaturation. However, these oligomers had a greatly reduced ability to refold substrate and did not respond to stimulation by DnaJ. Finally, we observed oligomeric DnaK in Escherichia coli cellular lysates by native gel electrophoresis and found that these structures became noticeably more prevalent in cells exposed to heat shock. Together, these studies suggest that DnaK oligomers are composed of ordered multimers that are functionally distinct from monomeric DnaK. Thus, oligomerization of DnaK might be an important step in chaperone cycling.

Rhodacyanine derivative selectively targets cancer cells and overcomes tamoxifen resistance.

Abstract: MKT-077, a rhodacyanine dye, was shown to produce cancer specific cell death. However, complications prevented the use of this compound beyond clinical trials. Here we describe YM-1, a derivative of MKT-077. We found that YM-1 was more cytotoxic and localized differently than MKT-077. YM-1 demonstrated this cytotoxicity across multiple cancer cell lines. This toxicity was limited to cancer cell lines; immortalized cell models were unaffected. Brief applications of YM-1 were found to be non-toxic. Brief treatment with YM-1 restored tamoxifen sensitivity to a refractory tamoxifen-resistant MCF7 cell model. This effect is potentially due to altered estrogen receptor alpha phosphorylation, an outcome precipitated by selective reductions in Akt levels (Akt/PKB). Thus, modifications to the rhodocyanine scaffold could potentially be made to improve efficacy and pharmacokinetic properties. Moreover, the impact on tamoxifen sensitivity could be a new utility for this compound family.

Molecular chaperones DnaK and DnaJ share predicted binding sites on most proteins in the E. coli proteome

Abstract: In Escherichia coli, the molecular chaperones DnaK and DnaJ cooperate to assist the folding of newly synthesized or unfolded polypeptides. DnaK and DnaJ bind to hydrophobic motifs in these proteins and they also bind to each other. Together, this system is thought to be sufficiently versatile to act on the entire proteome, which creates interesting challenges in understanding the interactions between DnaK, DnaJ and their thousands of potential substrates. To address this question, we computationally predicted the number and frequency of DnaK- and DnaJ-binding motifs in the E. coli proteome, guided by free energy-based binding consensus motifs. This analysis revealed that nearly every protein is predicted to contain multiple DnaK- and DnaJ-binding sites, with the DnaJ sites occurring approximately twice as often. Further, we found that an overwhelming majority of the DnaK sites partially or completely overlapped with the DnaJ-binding motifs. It is well known that high concentrations of DnaJ inhibit DnaK–DnaJ-mediated refolding. The observed overlapping binding sites suggest that this phenomenon may be explained by an important balance in the relative stoichiometry of DnaK and DnaJ. To test this idea, we measured the chaperone-assisted folding of two denatured substrates and found that the distribution of predicted DnaK- and DnaJ-binding sites was indeed a good predictor of the optimal stoichiometry required for folding. These studies provide insight into how DnaK and DnaJ might cooperate to maintain global protein homeostasis.

Pharmacological tuning of heat shock protein 70 modulates polyglutamine toxicity and aggregation.

Abstract: Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.

Inducible Renitence Limits Listeria monocytogenes Escape from Vacuoles in Macrophages

Abstract: Membranes of endolysosomal compartments in macrophages are often damaged by physical or chemical effects of particles ingested through phagocytosis or by toxins secreted by intracellular pathogens. This study identified a novel inducible activity in macrophages that increases resistance of phagosomes, late endosomes, and lysosomes to membrane damage. Pretreatment of murine macrophages with LPS, peptidoglycan, TNF-α, or IFN-γ conferred protection against subsequent damage to intracellular membranes caused by photooxidative chemistries or by phagocytosis of ground silica or silica microspheres. Phagolysosome damage was partially dependent on reactive oxygen species but was independent of the phagocyte oxidase. IFN-γ-stimulated macrophages from mice lacking the phagocyte oxidase inhibited escape from vacuoles by the intracellular pathogen Listeria monocytogenes, which suggested a role for this inducible renitence (resistance to pressure) in macrophage resistance to infection by pathogens that damage intracellular membranes. Renitence and inhibition of L. monocytogenes escape were partially attributable to heat shock protein-70. Thus, renitence is a novel, inducible activity of macrophages that maintains or restores the integrity of endolysosomal membranes.

Synthetic lethal interactions in yeast reveal functional roles of J protein co-chaperones

Abstract: J proteins are a diverse family of co-chaperones that cooperate with heat shock protein 70 (Hsp70) to coordinate protein quality control, especially in response to cellular stress. Current models suggest that individual J proteins might play roles in recruiting Hsp70s to specific functions, such as maintaining cell wall integrity or promoting ribosome biogenesis. However, relatively few stresses have been used to test this model and, as a result, only a few specific activities have been identified. To expand our understanding of the J protein network, we used a synthetic lethal approach in which 11 Saccharomyces cerevisiae deletion strains were treated with 12 well-characterized chemical inhibitors. The results defined new roles for specific J proteins in major signaling pathways. For example, an important role for Swa2 in cell wall integrity was identified and activities of the under-explored Jjj1, Apj1, Jjj3 and Caj1 proteins were suggested. More generally, these findings support a model in which some J proteins, such as Ydj1 and Zuo1, play “generalist” roles, while others, such as Apj1 and Jjj2, are “specialists”, having roles in relatively few pathways. Together, these results provide new insight into the network of J proteins.

The three cornerstones of chemical biology: innovative probes, new discoveries, and enabling tools.

Abstract: New Discoveries, and Enabling Tools Andrea D. Thompson,† Leah N. Makley,† Kathryn McMenimen,‡ and Jason E. Gestwicki*,† †Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States ‡Department of Chemistry, Mt. Holyoke College, South Hadley, Massachusetts 01075, United States K Symposia have a long history of serving as catalysts for the advancement of biomedical and life sciences by connecting scientists within and across disciplines. Thus, it was a natural extension of the central Keystone mission to hold the first ever Keystone Symposia on “Chemical Biology and Novel Tools in Pharmacology” on February 12−16, 2012. Organized by Laura Kiessling (University of Wisconsin, Madison), Jason Gestwicki (University of Michigan), and Kevan Shokat (University of California, San Francisco), this conference brought scientists from the international chemical biology community together in Santa Fe, New Mexico to communicate new ideas and technologies as well as recent progress in research. Unlike the majority of Keystone Symposia, this meeting was not focused on a particular aspect of biology but rather nucleated chemists and biologists across a wide range of disease areas and biological topics, providing an especially vibrant forum for the discussion of cross-platform ideas. As evidence of the breadth of the community, representatives from 10 countries, including academic, industrial, and government researchers and more than 40 graduate students, came together for this meeting. The conference schedule was filled with impressive presentations from experts across multiple areas of chemical biology (Figure 1). Throughout the program’s various sessions, three overarching themes emerged: (A) Design and Discovery of New Molecules and Probes, (B) New Advances in Biology Identified Using Chemical Approaches, and (C) Development of Novel, Enabling Methods (Figure 2). Select highlights are described here to illustrate the breadth and depth of these concepts.