Polyanions provide selective control of APC/C interactions with the activator subunit

Transient interactions between the Anaphase-Promoting Complex/Cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates

Genetically Engineered Microvesicles Carrying Suicide mRNA/Protein Inhibit Schwannoma Tumor Growth

Microvesicles (MVs) play an important role in intercellular communication by carrying mRNAs, microRNAs (miRNAs), non-coding RNAs, proteins, and DNA from cell to cell. To our knowledge, this is the first report of delivery of a therapeutic mRNA/protein via MVs for treatment of cancer. We first generated genetically engineered MVs by expressing high levels of the suicide gene mRNA and protein–cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT) in MV donor cells. MVs were isolated from these cells and used to treat pre-established nerve sheath tumors (schwannomas) in an orthotopic mouse model. We demonstrated that MV-mediated delivery of CD-UPRT mRNA/protein by direct injection into schwannomas led to regression of these tumors upon systemic treatment with the prodrug (5-fluorocytosine (5-FC)), which is converted within tumor cells to 5-fluorouracil (5-FU)–an anticancer agent. Taken together, these studies suggest that MVs can serve as novel cell-derived “liposomes” to effectively deliver therapeutic mRNA/proteins to treatment of diseases

Polyanions provide selective control of APC/C interactions with the activator subunit

Transient interactions between the Anaphase-Promoting Complex/Cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates

Mrx6 regulates mitochondrial DNA copy number in Saccharomyces cerevisiae by engaging the evolutionarily conserved Lon protease Pim1.

Mitochondrial function depends crucially on the maintenance of multiple mitochondrial DNA (mtDNA) copies. Surprisingly, the cellular mechanisms regulating mtDNA copy number remain poorly understood. Through a systematic high-throughput approach in Saccharomyces cerevisiae, we determined mtDNA-to-nuclear DNA ratios in 5148 strains lacking nonessential genes. The screen revealed MRX6, a largely uncharacterized gene, whose deletion resulted in a marked increase in mtDNA levels, while maintaining wild type-like mitochondrial structure and cell size. Quantitative superresolution imaging revealed that deletion of MRX6 alters both the size and the spatial distribution of mtDNA nucleoids. We demonstrate that Mrx6 partially colocalizes with mtDNA within mitochondria and interacts with the conserved Lon protease Pim1 in a complex that also includes Mam33 and the Mrx6-related protein Pet20. Acute depletion of Pim1 phenocopied the high mtDNA levels observed in Δmrx6 cells. No further increase in mtDNA copy number was observed upon depletion of Pim1 in Δmrx6 cells, revealing an epistatic relationship between Pim1 and Mrx6. Human and bacterial Lon proteases regulate DNA replication by degrading replication initiation factors, suggesting a model in which Pim1 acts similarly with the Mrx6 complex, providing a scaffold linking it to mtDNA

Additional file 1: of Quantitative framework for ordered degradation of APC/C substrates

Supplementary Figures. Figure S1. Ordered degradation of APC/C substrates. Figure S2. Determination of the time of degradation onset. Figure S3. Mechanisms that determine substrate degradation timing all change degradation onset. Figure S4. Ordinary differential equations for analysis of substrate degradation in the one-substrate model. Figure S5. APC/CCdc20 levels in the cell are lower than those of its substrates. Figure S6. Including deubiquitination in the model further limits the parameter space that generates a good delay in degradation onset and a fast rate of degradation. Figure S7. Changing k d influences T95 when not all APC/CCdc20 is bound to the substrate. Figure S8. Effects of doubling the concentration of C on T95 of S. (PDF 5283 kb