Optimization of a nicotine degrading enzyme for potential use in treatment of nicotine addiction.

BACKGROUND: Smoking and tobacco use continue to be the largest preventable causes of death globally. A novel therapeutic approach has recently been proposed: administration of an enzyme that degrades nicotine, the main addictive component of tobacco, minimizing brain exposure and reducing its reinforcing effects. Pre-clinical proof of concept has been previously established through dosing the amine oxidase NicA2 from Pseudomonas putida in rat nicotine self-administration models of addiction. RESULTS: This paper describes efforts towards optimizing NicA2 for potential therapeutic use: enhancing potency, improving its pharmacokinetic profile, and attenuating immunogenicity. Libraries randomizing residues located in all 22 active site positions of NicA2 were screened. 58 single mutations with 2- to 19-fold enhanced catalytic activity compared to wt at 10 µM nicotine were identified. A novel nicotine biosensor assay allowed efficient screening of the many primary hits for activity at nicotine concentrations typically found in smokers. 10 mutants with improved activity in rat serum at or below 250 nM were identified. These catalytic improvements translated to increased potency in vivo in the form of further lowering of nicotine blood levels and nicotine accumulation in the brains of Sprague-Dawley rats. Examination of the X-ray crystal structure suggests that these mutants may accelerate the rate limiting re-oxidation of the flavin adenine dinucleotide cofactor by enhancing molecular oxygen's access. PEGylation of NicA2 led to prolonged serum half-life and lowered immunogenicity observed in a human HLA DR4 transgenic mouse model, without impacting nicotine degrading activity. CONCLUSIONS: Systematic mutational analysis of the active site of the nicotine-degrading enzyme NicA2 has yielded 10 variants that increase the catalytic activity and its effects on nicotine distribution in vivo at nicotine plasma concentrations found in smokers. In addition, PEGylation substantially increases circulating half-life and reduces the enzyme's immunogenic potential. Taken together, these results provide a viable path towards generation of a drug candidate suitable for human therapeutic use in treating nicotine addiction.

Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II.

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3'-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions.

In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5'-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage.

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.

Simple enzymatic assays for the in vitro motor activity of transcription termination factor Rho from Escherichia coli.

The transcription termination factor Rho from Escherichia coli is a ring-shaped homo-hexameric protein that preferentially interacts with naked cytosine-rich Rut (Rho utilization) regions of nascent RNA transcripts. Once bound to the RNA chain, Rho uses ATP as an energy source to produce mechanical work and disruptive forces that ultimately lead to the dissociation of the ternary transcription complex. Although transcription termination assays have been useful to study Rho activity in various experimental contexts, they do not report directly on Rho mechanisms and kinetics. Here, we describe complementary ATP-dependent RNA-DNA helicase and streptavidin displacement assays that can be used to monitor in vitro Rho's motor activity in a more direct and quantitative manner.

Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.

Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro. Using pre-steady state kinetic analysis, we show that RPB9 deletion promotes sequestration of NTPs in the polymerase active center just prior to the phosphodiester bond formation. We propose a model in which the Rpb9 subunit controls transcription fidelity by delaying the closure of the trigger loop on the incoming NTP via interaction between the C-terminal domain of Rpb9 and the trigger loop. Our findings reveal a mechanism for regulation of transcription fidelity by protein factors located at a large distance from the active center of RNA polymerase II.

Noncanonical interactions in the management of RNA structural blocks by the transcription termination rho helicase.

To trigger transcription termination, the ring-shaped RNA-DNA helicase Rho from Escherichia coli chases the RNA polymerase along the nascent transcript, starting from a single-stranded C-rich Rut (Rho utilization) loading site. In some instances, a small hairpin structure divides harmlessly the C-rich loading region into two smaller Rut subsites, best exemplified by the tR1 terminator from phage lambda. Here, we show that the Rho helicase can also elude a RNA structural block located far downstream from the single-stranded C-rich region but upstream from a reporter RNA-DNA hybrid. In this process, Rho hexamers do not melt the intervening RNA motif but require single-stranded RNA segments on both of its sides. The reaction is also favored by physiological glutamate ions and can implicate Rho primary recognition of 5'-YC dimers (as for Rut binding) significantly upstream (>70 nucleotides) from the intervening motif. Surprisingly, we also found that primary interactions of Rho with 2'-hydroxyl groups located upstream from the intervening RNA structure serve to elude the motif. This demonstrates that the preference of Rho for RNA residues is not limited to the secondary interaction site that mediates ATPase-fuelled mechanochemistry within the hexamer central channel. These features could be part of an energy-effective mechanism in which Brownian exploration of the conformation of the Rho-substrate complex and accommodation of downstream secondary structures within a composite primary interaction site replace ATP-dependent translocation of the Rho enzyme along corresponding structured portions of the RNA chain.

Testing the steric exclusion model for hexameric helicases: substrate features that alter RNA-DNA unwinding by the transcription termination factor Rho.

Typical hexameric helicases form ring-shaped structures involved in DNA replication. These enzymes have been proposed to melt forked DNA substrates by binding to, and pulling, one strand within their central channel, while the other strand is forced outside of the hexamer by steric exclusion and specific contacts with the outer ring surface. Transcription termination factor Rho also assembles into ring-shaped hexamers that are capable to use NTP-derived energy to unwind RNA and RNA-DNA helices. To delineate the potential relationship between helicase structural organization and unwinding mechanism, we have performed in vitro Rho helicase experiments with model substrates containing an RNA-DNA helix downstream from a Rho loading site. We show that a physical discontinuity (nick) inhibits RNA-DNA unwinding when present in the RNA but not in the DNA strand. Moreover, the presence of a 3'-overhanging DNA tail (Y-shaped substrate) does not affect initial Rho binding but can impair helicase activity. This inhibitory effect varies with the length of the tail, is independent of the identity (A or U) of the tail residues, and is also obtained when a biotin-streptavidin complex replaces the single-stranded DNA arm. However, it is readily relaxed upon moving the reporter RNA-DNA helix farther from the Rho loading site. The data indicate that the Rho helicase uses a steric exclusion mechanism whereby the initial formation of a productive Rho-transcript complex is a crucial rate-limiting event, while no specific interactions with the displaced strand are required. These results outline significant similarities as well as some differences in the mechanism of unwinding between Rho and other hexameric helicases which are discussed in relation with the biological function of the Rho helicase.

Influence of substrate composition on the helicase activity of transcription termination factor Rho: reduced processivity of Rho hexamers during unwinding of RNA-DNA hybrid regions.

Transcription termination factor Rho forms ring-shaped hexameric structures that load onto segments of the nascent RNA transcript that are C-rich and mostly single-stranded. This interaction converts Rho hexamers into active molecular motors that use the energy resulting from their ATP hydrolase activity to move towards the transcript 3'-end. Upon translocation along the RNA chain, Rho can displace physical roadblocks, such as those formed by RNA-DNA helices, a feature that is likely central to the transcription termination mechanism. To study this "translocase" (helicase) activity, we have designed a collection of Rho substrate chimeras containing an RNA-DNA helix located at various positions with respect to a short (47 nucleotides) artificial loading site. We show that these synthetic constructs represent interesting model substrates able to engage in a productive interaction with Rho and to direct NTP-dependent [5'-->3']-translocation of the hexamers. Using both single and multiple-cycle experimental set-ups, we have also found that Rho helicase activity is strongly dependent on the substrate composition and reaction conditions. For this reason, the rate-limiting step of the helicase reaction could not be identified unambiguously. Yet, the linear dependence of the reaction rate on the hybrid length suggests that helicase action on the RNA-DNA region could be controlled by a unique slow step such as Rho activation, conformational rearrangement, or DNA release. Moreover, removal of the DNA strand occurred at a significant cost for the Rho enzyme, inducing, on average, dissociation from the substrate for every 60-80 base-pairs of hybrid unwound. These results are discussed in relation to the known requirements for Rho substrates, general features of hexameric helicases, and current models for Rho-dependent transcription termination.