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Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

ABSTRACT: RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR?+?RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

SUBMITTER: Sinapah S

PROVIDER: S-EPMC3035440 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

Sinapah Sylvie S Wu Shiying S Chen Yu Y Pettersson B M Fredrik BM Gopalan Venkat V Kirsebom Leif A LA

Nucleic acids research 20101008 3

RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts ...[more]

PMID: 20935047

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Project description:Mutations in amyloid ? precursor protein (APP) gene alter APP processing, either causing familial Alzheimer's disease (AD) or protecting against dementia. Under normal conditions, ?-site APP cleaving enzyme 1 (BACE1) cleaves APP at minor Asp1 site to generate C99 for amyloid ? protein (A?) production, and predominantly at major Glu11 site to generate C89, resulting in truncated A? production. We discovered that A673V mutation, the only recessive AD-associated APP mutation, shifted the preferential ?-cleavage site of BACE1 in APP from the Glu11 site to the Asp1 site both in male and female transgenic mice in vivo and in cell lines and primary neuronal culture derived from timed pregnant rats in vitro, resulting in a much higher C99 level and C99/C89 ratio. All other mutations at this site, including the protective Icelandic A673T mutation, reduced C99 generation, and decreased the C99/C89 ratio. Furthermore, A673V mutation caused stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited ?-secretase cleavage of the mutant C99 to generate A?, leading to recessively inherited AD. The results demonstrate that APP673 regulates APP processing and the BACE1 cleavage site selection is critical for amyloidogenesis in AD pathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development.SIGNIFICANCE STATEMENT ?-site APP cleaving enzyme 1 (BACE1) is essential for amyloid ? protein production. We discovered that A673V mutation shifted the BACE1 cleavage site from the Glu11 to the Asp1 site, resulting in much higher C99 level and C99/C89 ratio. All other mutations at this site of amyloid ? precursor protein (APP) reduced C99 generation and decreased the C99/C89 ratio. Furthermore, A673V mutation resulted in stronger dimerization between mutant and wild-type APP, enhanced the lysosomal degradation of the mutant APP, and inhibited ?-secretase cleavage of the mutant C99 to generate amyloid ? protein, leading to recessively inherited Alzheimer's disease (AD). The results demonstrate that APP673 regulates APP processing, and the BACE1 cleavage site selection is critical for amyloidogenesis in AD pathogenesis, and implicate a pharmaceutical potential for targeting the APP673 site for AD drug development.

Dataset Information

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

Publications

Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

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