Self-oligomerization Regulates Stability of Survival Motor Neuron Protein Isoforms by Sequestering an SCF\u3csup\u3eSlmb\u3c/sup\u3e Degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

The group II intron ribonucleoprotein precursor is a large, loosely packed structure

Group II self-splicing introns are phylogenetically diverse retroelements that are widely held to be the ancestors of spliceosomal introns and retrotransposons that insert into DNA. Folding of group II intron RNA is often guided by an intron-encoded protein to form a catalytically active ribonucleoprotein (RNP) complex that plays a key role in the activity of the intron. To date, possible structural differences between the intron RNP in its precursor and spliced forms remain unexplored. In this work, we have trapped the native Lactococcus lactis group II intron RNP complex in its precursor form, by deleting the adenosine nucleophile that initiates splicing. Sedimentation velocity, size-exclusion chromatography and cryo-electron microscopy provide the first glimpse of the intron RNP precursor as a large, loosely packed structure. The dimensions contrast with those of compact spliced introns, implying that the RNP undergoes a dramatic conformational change to achieve the catalytically active state

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Structural insights into the mechanism and inhibition of prostaglandin H2 synthase-1

Prostanoids are fatty acid autocoids that are involved in platelet aggregation, inflammation, and the pathophysiology of cardiovascular disease. Prostaglandin H2 synthase (PGHS; EC 1.14.99.1) is an important drug target essential to their formation. This bifunctional heme-dependent enzyme functions as a homodimer, where each monomer contains both a cyclooxygenase and a peroxidase active site. These active sites work in concert to transform arachidonic acid into PGH2, the precursor to the prostanoids. There are two known isoforms of this enzyme, which differ mainly in their expression patterns: PGHS-1 is a constitutively expressed isoform involved in “housekeeping” functions, while PGHS-2 expression is induced in response to inflammatory stimuli. Both isoforms are susceptible to inhibition by the class of drugs known as nonsteroidal antiinflammatory drugs (NSAIDs), which act by blocking the cyclooxygenase active site. The goal of this dissertation was to use x-ray crystallography to expand the current understanding of the structure of PGHS. In doing so, key issues, including the structural consequences of peroxidase impairment by protoporphyrin substitution and the structural basis of NSAID binding were investigated. As a result of this study, six unique x-ray crystal structures of PGHS were determined. Among these structures, the first high-resolution (2.0 Å) structures of PGHS-1 were solved, and together these structures yield insights into the architecture of the two active sites of PGHS, the nature of inhibitor binding in the cyclooxygenase active site, and the structural consequences of protoporphyrin substitution

Structural insights into the mechanism and inhibition of prostaglandin H2 synthase-1

Prostanoids are fatty acid autocoids that are involved in platelet aggregation, inflammation, and the pathophysiology of cardiovascular disease. Prostaglandin H2 synthase (PGHS; EC 1.14.99.1) is an important drug target essential to their formation. This bifunctional heme-dependent enzyme functions as a homodimer, where each monomer contains both a cyclooxygenase and a peroxidase active site. These active sites work in concert to transform arachidonic acid into PGH2, the precursor to the prostanoids. There are two known isoforms of this enzyme, which differ mainly in their expression patterns: PGHS-1 is a constitutively expressed isoform involved in “housekeeping” functions, while PGHS-2 expression is induced in response to inflammatory stimuli. Both isoforms are susceptible to inhibition by the class of drugs known as nonsteroidal antiinflammatory drugs (NSAIDs), which act by blocking the cyclooxygenase active site. The goal of this dissertation was to use x-ray crystallography to expand the current understanding of the structure of PGHS. In doing so, key issues, including the structural consequences of peroxidase impairment by protoporphyrin substitution and the structural basis of NSAID binding were investigated. As a result of this study, six unique x-ray crystal structures of PGHS were determined. Among these structures, the first high-resolution (2.0 Å) structures of PGHS-1 were solved, and together these structures yield insights into the architecture of the two active sites of PGHS, the nature of inhibitor binding in the cyclooxygenase active site, and the structural consequences of protoporphyrin substitution