Brian P. Callahan

Modulation of intein activity by its neighboring extein substrates

Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N- and C-terminal exteins can accelerate the N-terminal cleavage rate by >4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.

Cisplatin inhibits protein splicing suggesting inteins as therapeutic targets in mycobacteria

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC50 of ∼2 μm, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ∼40 μm, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.

Zinc Inhibits Hedgehog Autoprocessing LINKING ZINC DEFICIENCY WITH HEDGEHOG ACTIVATION

Background: In many types of cancers zinc deficiency and overproduction of Hedgehog (Hh) ligand co-exist. Results: Zinc binds to the active site of the Hedgehog-intein (Hint) domain and inhibits Hh ligand production both in vitro and in cell culture. Conclusion: Zinc influences the Hh autoprocessing. Significance: This study uncovers a novel mechanistic link between zinc and the Hh signaling pathway. Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand. In an in vitro Hh autoprocessing assay we show that zinc inhibits Hh autoprocessing with a Ki of 2 μm. We then demonstrate that zinc inhibits Hh autoprocessing in a cellular environment with experiments in primary rat astrocyte culture. Solution NMR reveals that zinc binds the active site residues of the Hh autoprocessing domain to inhibit autoprocessing, and isothermal titration calorimetry provided the thermodynamics of the binding. In normal physiology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, ovarian cancer, and autism. Our data suggest a causal relationship between zinc deficiency and the overproduction of Hh ligand.

Active Site Targeting of Hedgehog Precursor Protein with Phenylarsine Oxide

Hedgehog proteins, signaling molecules implicated in human embryo development and cancer, can be inhibited at the stage of autoprocessing by the trivalent arsenical phenyl arsine oxide (PhAsIII). The interaction (apparent Ki, 4×10−7 M) is characterized by an optical binding assay and by NMR spectroscopy. PhAsIII appears to be the first validated inhibitor of hedgehog autoprocessing, which is unique to hedgehog proteins and essential for biological activity.

Hedgehog Cholesterolysis: Specialized Gatekeeper to Oncogenic Signaling

Discussions of therapeutic suppression of hedgehog (Hh) signaling almost exclusively focus on receptor antagonism; however, hedgehog’s biosynthesis represents a unique and potentially targetable aspect of this oncogenic signaling pathway. Here, we review a key biosynthetic step called cholesterolysis from the perspectives of structure/function and small molecule inhibition. Cholesterolysis, also called cholesteroylation, generates cholesterol-modified Hh ligand via autoprocessing of a hedgehog precursor protein. Post-translational modification by cholesterol appears to be restricted to proteins in the hedgehog family. The transformation is essential for Hh biological activity and upstream of signaling events. Despite its decisive role in generating ligand, cholesterolysis remains conspicuously unexplored as a therapeutic target.

1H, 13C, and 15N NMR assignments of a Drosophila Hedgehog autoprocessing domain

The Hedgehog (Hh) signaling pathway plays important roles in embryonic growth and patterning in different organisms. Abnormal activity of the Hh signaling pathway has been associated to cancers, holoprosencephaly and autism spectrum disorders. The backbone and side chain resonance assignments of a Drosophila Hh autoprocessing domain have been determined based on triple-resonance experiments with the [13C, 15N]-labeled and [2H, 13C, 15N])-labeled proteins.

A Single Aspartate Coordinates Two Catalytic Steps in Hedgehog Autoprocessing.

Hedgehog (Hh) signaling is driven by the cholesterol-modified Hh ligand, generated by autoprocessing of Hh precursor protein. Two steps in Hh autoprocessing, N-S acyl shift and transesterification, must be coupled for efficient Hh cholesteroylation and downstream signal transduction. In the present study, we show that a conserved aspartate residue, D46 of the Hh autoprocessing domain, coordinates these two catalytic steps. Mutagenesis demonstrated that D46 suppresses non-native Hh precursor autoprocessing and is indispensable for transesterification with cholesterol. NMR measurements indicated that D46 has a pKa of 5.6, ∼2 units above the expected pKa of aspartate, due to a hydrogen-bond between protonated D46 and a catalytic cysteine residue. However, the deprotonated form of D46 side chain is also essential, because a D46N mutation cannot mediate cholesteroylation. On the basis of these data, we propose that the proton shuttling of D46 side chain mechanistically couples the two steps of Hh cholesteroylation.

Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer.

Abiraterone, a potent inhibitor of the human enzyme CYP17A1 (cytochrome P450c17), provides a last line of defense against ectopic androgenesis in advanced prostate cancer. Herein we report an unprecedented off‐target interaction between abiraterone and oncogenic hedgehog proteins. Our experiments indicate that abiraterone and its structural congener, galeterone, can replace cholesterol as a substrate in a specialized biosynthetic event of hedgehog proteins, known as cholesterolysis. The off‐target reaction generates covalent hedgehog–drug conjugates. Cell‐based reporter assays indicate that these conjugates activate hedgehog signaling when present in the low nanomolar range. Because hedgehog signaling is implicated in prostate cancer progression, and abiraterone is administered to treat advanced stages of the disease, this off‐target interaction may have therapeutic significance.

Chemical Bypass of General Base Catalysis in Hedgehog Protein Cholesterolysis Using a Hyper-Nucleophilic Substrate

Proteins in the hedgehog family undergo self-catalyzed endoproteolysis involving nucleophilic attack by a molecule of cholesterol. Recently, a conserved aspartate residue (D303, or D46) of hedgehog was identified as the general base that activates cholesterol during this unusual autoprocessing event; mutation of the catalyzing functional group (D303A) reduces activity by >104-fold. Here we report near total rescue of this ostensibly dead general base mutant by a synthetic substrate, 3β-hydroperoxycholestane (3HPC) in which the sterol -OH group is replaced by the hyper nucleophilic -OOH group. Other hedgehog point mutants at D303, also unreactive with cholesterol, accepted 3HPC as a substrate with the rank order: WT > D303A ≈ D303N ≫ D303R, D303E. We attribute the revived activity with 3-HPC to the α-effect, where tandem electronegative atoms exhibit exceptionally high nucleophilicity despite relatively low basicity.