Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients.

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998-1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co-chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90-mediated folding of kinase and non-kinase clients-including Tsc2-thereby preventing their ubiquitination and proteasomal degradation.

RPtag as an Orally Bioavailable, Hyperstable Epitope Tag and Generalizable Protein Binding Scaffold.

Antibodies are the most prolific biologics in research and clinical environments because of their ability to bind targets with high affinity and specificity. However, antibodies also carry liabilities. A significant portion of the life-science reproducibility crisis is driven by inconsistent performance of research-grade antibodies, and clinical antibodies are often unstable and require costly cold-chain management to reach their destinations in active form. In biotechnology, antibodies are also limited by difficulty integrating them in many recombinant systems due to their size and structural complexity. A switch to small, stable, sequence-verified binding scaffolds may overcome these barriers. Here we present such a scaffold, RPtag, based on a ribose-binding protein (RBP) from extremophile Caldanaerobacter subterraneus. RPtag binds an optimized peptide with pM affinity, is stable to extreme temperature, pH, and protease treatment, readily refolds after denaturation, is effective in common laboratory applications, was rationally engineered to bind bioactive PDGF-ß, and was formulated as a gut-stable orally bioavailable preparation.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore.

p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.

Rare Sighting of Posterior Communicating Artery Infundibulum With Posterior Cerebral Artery Origin.

Infundibula are funnel-shaped lesions that develop at the intersections of major intracranial arteries. These lesions are prone to being misdiagnosed as intracranial aneurysms. The most common arterial infundibula have been noted in the posterior communicating artery (PCoA) branch of the internal carotid artery (ICA). Digitally subtracted angiography performed included catheter angiography of the vertebral artery and ipsilateral carotid to evaluate the suspected lesion. Right vertebral angiography demonstrated an infundibulum seen at the right PCoA/posterior cerebral artery (PCA) junction, with noted posterior-to-anterior circulation dominance of the Circle of Willis collateral flow. We report a case of posterior communicating artery infundibulum arising from the posterior cerebral artery origin in a 38-year-old man.

Catalytic inhibitor of Protein Phosphatase 5 activates the extrinsic apoptotic pathway by disrupting complex II in kidney cancer.

Serine/threonine protein phosphatase-5 (PP5) is involved in tumor progression and survival, making it an attractive therapeutic target. Specific inhibition of protein phosphatases has remained challenging because of their conserved catalytic sites. PP5 contains its regulatory domains within a single polypeptide chain, making it a more desirable target. Here we used an in silico approach to screen and develop a selective inhibitor of PP5. Compound P053 is a competitive inhibitor of PP5 that binds to its catalytic domain and causes apoptosis in renal cancer. We further demonstrated that PP5 interacts with FADD, RIPK1, and caspase 8, components of the extrinsic apoptotic pathway complex II. Specifically, PP5 dephosphorylates and inactivates the death effector protein FADD, preserving complex II integrity and regulating extrinsic apoptosis. Our data suggests that PP5 promotes renal cancer survival by suppressing the extrinsic apoptotic pathway. Pharmacologic inhibition of PP5 activates this pathway, presenting a viable therapeutic strategy for renal cancer.

4-aminophenylalanine as a biocompatible nucleophilic catalyst for hydrazone ligations at low temperature and neutral pH.

Hydrazone formation and similar reactions are highly versatile and specific, but their application to biological systems has been limited by their characteristically slow reaction kinetics at neutral pH. Catalysis of these reactions through imine formation with aromatic amines such as aniline has broadened the applicability of these reactions to biomolecular labeling. High concentrations of the catalyst are necessary, which may be incompatible with the native structure of certain proteins. In this study, we investigated the utility of 4-aminophenylalanine (4a-Phe) as a catalyst for these reactions. We find that 4a-Phe is nearly as effective as aniline in catalyzing hydrazone formation between the reactive amino acid 3-formyltyrosine (3f-Tyr) and hydrazine-containing fluorophores, both free in solution and incorporated into the protein tubulin. The catalyst 4a-Phe maintains ∼70% of the catalytic efficacy of aniline and is less detrimental to the native structure of tubulin. Examination of the temperature dependence of imine formation between 3f-Tyr and 4a-Phe shows an increase in imine concentration accompanying a decrease in temperature, confirming the exothermic nature of the equilibrium reaction. Interestingly, decreasing the temperature of the 4a-Phe-catalyzed hydrazone reaction between 3f-Tyr and the fluorophore 7-hydrazinyl-4-methylcoumarin increases the overall rate of the reaction. This result indicates that the temperature dependence of the catalyst-aldehyde equilibrium is greater than the temperature dependence of the rate constant for hydrazone formation from this intermediate, and that the rate of hydrazone formation a direct function of the concentration of the intermediate imine. These results provide a platform for conducting nucleophilic catalysis under conditions that are more compatible with biomolecular targets than previously demonstrated, thereby expanding the utility of hydrazone ligations in biological systems.

Design, synthesis and biological evaluation of a simplified fluorescently labeled discodermolide as a molecular probe to study the binding of discodermolide to tubulin.

The design, synthesis, and biological evaluation of a simplified fluorescently labeled discodermolide analogue possessing a dimethylaminobenzoyl fluorophore has been achieved. Stereoselective Suzuki coupling and Horner-Wadsworth-Emmons reaction comprised the key tactics for its construction. The analogue exhibited qualitatively similar activity to paclitaxel in a tubulin assembly assay, and it can thus be used as a fluorescent molecular probe to explore the local environment of the discodermolide binding site on tubulin. The results of fluorescence measurements on the tubulin-bound analogue are reported.

Benzothiazolyl and Benzoxazolyl Hydrazones Function as Zinc Metallochaperones to Reactivate Mutant p53.

We identified a set of thiosemicarbazone (TSC) metal ion chelators that reactivate specific zinc-deficient p53 mutants using a mechanism called zinc metallochaperones (ZMCs) that restore zinc binding by shuttling zinc into cells. We defined biophysical and cellular assays necessary for structure-activity relationship studies using this mechanism. We investigated an alternative class of zinc scaffolds that differ from TSCs by substitution of the thiocarbamoyl moiety with benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones. Members of this series bound zinc with similar affinity and functioned to reactivate mutant p53 comparable to the TSCs. Acute toxicity and efficacy assays in rodents demonstrated C1 to be significantly less toxic than the TSCs while demonstrating equivalent growth inhibition. We identified C85 as a ZMC with diminished copper binding that functions as a chemotherapy and radiation sensitizer. We conclude that the benzothiazolyl, benzoxazolyl, and benzimidazolyl hydrazones can function as ZMCs to reactivate mutant p53 in vitro and in vivo.

The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect.

Aerobic glycolysis in cancer cells, also known as the 'Warburg effect', is driven by hyperactivity of lactate dehydrogenase A (LDHA). LDHA is thought to be a substrate-regulated enzyme, but it is unclear whether a dedicated intracellular protein also regulates its activity. Here, we identify the human tumor suppressor folliculin (FLCN) as a binding partner and uncompetitive inhibitor of LDHA. A flexible loop within the amino terminus of FLCN controls movement of the LDHA active-site loop, tightly regulating its enzyme activity and, consequently, metabolic homeostasis in normal cells. Cancer cells that experience the Warburg effect show FLCN dissociation from LDHA. Treatment of these cells with a decapeptide derived from the FLCN loop region causes cell death. Our data suggest that the glycolytic shift of cancer cells is the result of FLCN inactivation or dissociation from LDHA. Together, FLCN-mediated inhibition of LDHA provides a new paradigm for the regulation of glycolysis.

Characterization of the colchicine binding site on avian tubulin isotype betaVI.

Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 10(5) M(-1) for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb isotype betaVI is almost identical to that of its human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells.

Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutants.

Missense mutations in the p53 DNA-binding domain (DBD) contribute to half of new cancer cases annually. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. We find that DBD possesses two unusual properties-one of the highest zinc affinities of any eukaryotic protein and extreme instability in the absence of zinc-which are predicted to poise p53 on the cusp of folding/unfolding in the cell, with a major determinant being available zinc concentration. We analyze the 20 most common tumorigenic p53 mutations and find that 80% impair zinc affinity, thermodynamic stability, or both. Biophysical, cell-based, and murine xenograft experiments demonstrate that a synthetic zinc metallochaperone rescues not only mutations that decrease zinc affinity, but also mutations that destabilize DBD without impairing zinc binding. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients annually in the U.S.

Combinatorial Therapy of Zinc Metallochaperones with Mutant p53 Reactivation and Diminished Copper Binding.

Chemotherapy and radiation are more effective in wild-type (WT) p53 tumors due to p53 activation. This is one rationale for developing drugs that reactivate mutant p53 to synergize with chemotherapy and radiation. Zinc metallochaperones (ZMC) are a new class of mutant p53 reactivators that restore WT structure and function to zinc-deficient p53 mutants. We hypothesized that the thiosemicarbazone, ZMC1, would synergize with chemotherapy and radiation. Surprisingly, this was not found. We explored the mechanism of this and found the reactive oxygen species (ROS) activity of ZMC1 negates the signal on p53 that is generated with chemotherapy and radiation. We hypothesized that a zinc scaffold generating less ROS would synergize with chemotherapy and radiation. The ROS effect of ZMC1 is generated by its chelation of redox active copper. ZMC1 copper binding (K Cu) studies reveal its affinity for copper is approximately 108 greater than Zn2+ We identified an alternative zinc scaffold (nitrilotriacetic acid) and synthesized derivatives to improve cell permeability. These compounds bind zinc in the same range as ZMC1 but bound copper much less avidly (106- to 107-fold lower) and induced less ROS. These compounds were synergistic with chemotherapy and radiation by inducing p53 signaling events on mutant p53. We explored other combinations with ZMC1 based on its mechanism of action and demonstrate that ZMC1 is synergistic with MDM2 antagonists, BCL2 antagonists, and molecules that deplete cellular reducing agents. We have identified an optimal Cu2+:Zn2+ binding ratio to facilitate development of ZMCs as chemotherapy and radiation sensitizers. Although ZMC1 is not synergistic with chemotherapy and radiation, it is synergistic with a number of other targeted agents.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.

Quantification of Membrane Protein-Detergent Complex Interactions.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding.

Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes involved in maintaining the stability and activity of numerous signalling proteins, also known as clients. Hsp90 ATPase activity is essential for its chaperone function and it is regulated by co-chaperones. Here we show that the tumour suppressor FLCN is an Hsp90 client protein and its binding partners FNIP1/FNIP2 function as co-chaperones. FNIPs decelerate the chaperone cycle, facilitating FLCN interaction with Hsp90, consequently ensuring FLCN stability. FNIPs compete with the activating co-chaperone Aha1 for binding to Hsp90, thereby providing a reciprocal regulatory mechanism for chaperoning of client proteins. Lastly, downregulation of FNIPs desensitizes cancer cells to Hsp90 inhibitors, whereas FNIPs overexpression in renal tumours compared with adjacent normal tissues correlates with enhanced binding of Hsp90 to its inhibitors. Our findings suggest that FNIPs expression can potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors.

Reactivating mutant p53 using small molecules as zinc metallochaperones: awakening a sleeping giant in cancer.

Tumor protein p53 (TP53) is the most commonly mutated gene in human cancer. The majority of mutations are missense, and generate a defective protein that is druggable. Yet, for decades, the small-molecule restoration of wild-type (WT) p53 function in mutant p53 tumors (so-called p53 mutant 'reactivation') has been elusive to researchers. The p53 protein requires the binding of a single zinc ion for proper folding, and impairing zinc binding is a major mechanism for loss of function in missense mutant p53. Here, we describe recent work defining a new class of drugs termed zinc metallochaperones that restore WT p53 structure and function by restoring Zn(2+) to Zn(2+)-deficient mutant p53.

Small molecule restoration of wildtype structure and function of mutant p53 using a novel zinc-metallochaperone based mechanism.

UNLABELLED: NSC319726 (ZMC1) is a small molecule that reactivates mutant p53 by restoration of WT structure/function to the most common p53 missense mutant (p53-R175H). We investigated the mechanism by which ZMC1 reactivates p53-R175H and provide evidence that ZMC1: 1) restores WT structure by functioning as a zinc-metallochaperone, providing an optimal concentration of zinc to facilitate proper folding; and 2) increases cellular reactive oxygen species that transactivate the newly conformed p53-R175H (via post-translational modifications), inducing an apoptotic program. We not only demonstrate that this zinc metallochaperone function is possessed by other zinc-binding small molecules, but that it can reactivate other p53 mutants with impaired zinc binding. This represents a novel mechanism for an anti-cancer drug and a new pathway to drug mutant p53. SIGNIFICANCE: We have elucidated a novel mechanism to restore wild-type structure/function to mutant p53 using small molecules functioning as zinc-metallochaperones. The pharmacologic delivery of a metal ion to restore proper folding of a mutant protein is unique to medicinal chemistry and represents a new pathway to drug mutant p53.

An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models.

While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening.