Jason E. Gestwicki

Potential therapeutic applications of autophagy

Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest as it is now recognized to be involved in various developmental processes and various diseases including cancer and neurodegeneration. Autophagy can function as a cytoprotective mechanism; however, it also has the capacity to cause cell death. A better understanding of autophagy is needed to allow its manipulation for therapeutic purposes, and new insights into the molecular mechanisms of autophagy are now leading to the discovery of exciting new potential drug targets.

Synthetic multivalent ligands in the exploration of cell-surface interactions

Processes such as cell-cell recognition and the initiation of signal transduction often depend on the formation of multiple receptor-ligand complexes at the cell surface. Synthetic multivalent ligands are unique probes of these complex cell-surface-binding events. Multivalent ligands can be used as inhibitors of receptor-ligand interactions or as activators of signal transduction pathways. Emerging from these complementary applications is insight into how cells exploit multivalent interactions to bind with increased avidity and specificity and how cell-surface receptor organization influences signaling and the cellular responses that result.

A Field of Myocardial-Endocardial NFAT Signaling Underlies Heart Valve Morphogenesis

The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.

Heat Shock Proteins 70 and 90 Inhibit Early Stages of Amyloid β-(1–42) Aggregation in Vitro

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid β (Aβ) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Aβ aggregation. These observations led to the hypothesis that chaperones might interact with Aβ and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Aβ has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Aβ aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Aβ self-assembly and that these chaperones are effective at substoichiometric concentrations (∼1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Aβ is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.

Solution conformation of wild-type E. coli Hsp70 (DnaK) chaperone complexed with ADP and substrate.

DnaK is the canonical Hsp70 molecular chaperone protein from Escherichia coli. Like other Hsp70s, DnaK comprises two main domains: a 44-kDa N-terminal nucleotide-binding domain (NBD) that contains ATPase activity, and a 25-kDa substrate-binding domain (SBD) that harbors the substrate-binding site. Here, we report an experimental structure for wild-type, full-length DnaK, complexed with the peptide NRLLLTG and with ADP. It was obtained in aqueous solution by using NMR residual dipolar coupling and spin labeling methods and is based on available crystal structures for the isolated NBD and SBD. By using dynamics methods, we determine that the NBD and SBD are loosely linked and can move in cones of ±35° with respect to each other. The linker region between the domains is a dynamic random coil. Nevertheless, an average structure can be defined. This structure places the SBD in close proximity of subdomain IA of the NBD and suggests that the SBD collides with the NBD at this area to establish allosteric communication.

Heat Shock Protein 70 (Hsp70) as an Emerging Drug Target

Heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed in response to stress. In this role, Hsp70 binds to its protein substrates and stabilize them against denaturation or aggregation until conditions improve.1 In addition to its functions during a stress response, Hsp70 has multiple responsibilities during normal growth; it assists in the folding of newly synthesized proteins,2, 3 the subcellular transport of proteins and vesicles,4 the formation and dissociation of complexes,5 and the degradation of unwanted proteins.6, 7 Thus, this chaperone broadly shapes protein homeostasis by controlling protein quality control and turnover during both normal and stress conditions.8 Consistent with these diverse activities, genetic and biochemical studies have implicated it in a range of diseases, including cancer, neurodegeneration, allograft rejection and infection. This review provides a brief review of Hsp70 structure and function and then explores some of the emerging opportunities (and challenges) for drug discovery. Hsp70 is Highly Conserved Members of the Hsp70 family are ubiquitously expressed and highly conserved; for example, the major Hsp70 from Escherichia coli, termed DnaK, is approximately 50% identical to human Hsp70s.9 Eukaryotes often express multiple Hsp70 family members with major isoforms found in all the cellular compartments: Hsp72 (HSPA1A) and heat shock cognate 70 (Hsc70/HSPA8) in the cytosol and nucleus, BiP (Grp78/HSPA5) in the endoplasmic reticulum and mtHsp70 (Grp75/mortalin/HSPA9) in mitochondria. Some of the functions of the cytosolic isoforms, Hsc70 and Hsp72, are thought to be redundant, but the transcription of Hsp72 is highly responsive to stress and Hsc70 is constitutively expressed. In the ER and mitochondria, the Hsp70 family members are thought to fulfill specific functions and have unique substrates, with BiP playing key roles in the folding and quality control of ER proteins and mtHsp70 being involved in the import and export of proteins from the mitochondria. For the purposes of this review, we will often use Hsp70 as a generic term to encompass the shared properties of the family members.

Inter-receptor communication through arrays of bacterial chemoreceptors

The sensing mechanisms of chemotactic bacteria allow them to respond sensitively to stimuli. Escherichia coli, for example, respond to changes in chemoattractant concentration of less than 10% over a range spanning six orders of magnitude. Sensitivity over this range depends on a nonlinear relationship between ligand concentration and output response. At low ligand concentrations, substantial amplification of the chemotactic signal is required; however, the mechanism responsible for this amplification remains unclear. Here we demonstrate that inter-receptor communication within a lattice acts to amplify and integrate sensory information. Synthetic multivalent ligands that interact through the low-abundance, galactose-sensing receptor Trg stabilize large clusters of chemoreceptors and markedly enhance signal output from these enforced clusters. On treatment with multivalent ligands, the response to the attractant serine is amplified by at least 100-fold. This amplification requires a full complement of chemoreceptors; deletion of the aspartate (Tar) or dipeptide (Tap) receptors diminishes the amplification of the serine response. These results demonstrate that the entire array is involved in sensing. This mode of information exchange has general implications for the processing of signals by cellular receptors.

Structure–activity Relationships of Amyloid Beta‐aggregation Inhibitors Based on Curcumin: Influence of Linker Length and Flexibility

Self‐assembly of amyloid beta into fibrillar plaques is characteristic of Alzheimer’s disease and oligomers of this peptide are believed to be involved in neurodegeneration. Natural organic dyes, such as congo red and curcumin, bind tightly to amyloid beta and, at higher concentrations, block its self‐assembly. The ability of these molecules to prevent amyloid accumulation has generated interest in understanding which of their structural features contribute to inhibitory potency. In general, amyloid beta ligands tend to be flat, planar molecules with substituted aromatic end groups; however, a comprehensive structure–activity study has not been reported. To better understand these ligands, we surveyed the effect of three prominent features on inhibition of amyloid aggregation: the presence of two aromatic end groups, the substitution pattern of these aromatics, and the length and flexibility of the linker region. We found that modification of any one of the modules has profound effects on activity. Further, we report that the optimal length of the linker lies within a surprisingly narrow regime (6–19 Å). These results offer insight into the key chemical features required for inhibiting amyloid beta aggregation. In turn, these findings help define the nature of the docking site for small molecules on the amyloid beta surface.

Chemical Manipulation of Hsp70 ATPase Activity Regulates Tau Stability

Alzheimers disease and other tauopathies have recently been clustered with a group of nervous system disorders termed protein misfolding diseases. The common element established between these disorders is their requirement for processing by the chaperone complex. It is now clear that the individual components of the chaperone system, such as Hsp70 and Hsp90, exist in an intricate signaling network that exerts pleiotropic effects on a host of substrates. Therefore, we have endeavored to identify new compounds that can specifically regulate individual components of the chaperone family. Here, we hypothesized that chemical manipulation of Hsp70 ATPase activity, a target that has not previously been pursued, could illuminate a new pathway toward chaperone-based therapies. Using a newly developed high-throughput screening system, we identified inhibitors and activators of Hsp70 enzymatic activity. Inhibitors led to rapid proteasome-dependent tau degradation in a cell-based model. Conversely, Hsp70 activators preserved tau levels in the same system. Hsp70 inhibition did not result in general protein degradation, nor did it induce a heat shock response. We also found that inhibiting Hsp70 ATPase activity after increasing its expression levels facilitated tau degradation at lower doses, suggesting that we can combine genetic and pharmacologic manipulation of Hsp70 to control the fate of bound substrates. Disease relevance of this strategy was further established when tau levels were rapidly and substantially reduced in brain tissue from tau transgenic mice. These findings reveal an entirely novel path toward therapeutic intervention of tauopathies by inhibition of the previously untargeted ATPase activity of Hsp70.

Critical role of acetylation in tau-mediated neurodegeneration and cognitive deficits

Tauopathies, including frontotemporal dementia (FTD) and Alzheimers disease (AD), are neurodegenerative diseases in which tau fibrils accumulate. Recent evidence supports soluble tau species as the major toxic species. How soluble tau accumulates and causes neurodegeneration remains unclear. Here we identify tau acetylation at Lys174 (K174) as an early change in AD brains and a critical determinant in tau homeostasis and toxicity in mice. The acetyl-mimicking mutant K174Q slows tau turnover and induces cognitive deficits in vivo. Acetyltransferase p300-induced tau acetylation is inhibited by salsalate and salicylate, which enhance tau turnover and reduce tau levels. In the PS19 transgenic mouse model of FTD, administration of salsalate after disease onset inhibited p300 activity, lowered levels of total tau and tau acetylated at K174, rescued tau-induced memory deficits and prevented hippocampal atrophy. The tau-lowering and protective effects of salsalate were diminished in neurons expressing K174Q tau. Targeting tau acetylation could be a new therapeutic strategy against human tauopathies.