Single subunit degradation of WIZ, a lenalidomide- and pomalidomide dependent substrate of E3 ubiquitin ligase CRL4^(CRBN)

Immunomodulators (IMiDs) are an effective class of drugs used to treat blood cancers. IMiDs are believed to work by recruiting protein targets containing a β-hairpin motif for ubiquitination by E3 ubiquitin ligase complexes composed of cereblon (CRBN), Cullin-4a (CUL4a), DNA Damage Binding protein-1 (DDB1), and Ring Box-1 (RBX1). The ubiquitinated protein is subsequently degraded by the proteasome. By characterizing the repertoire of proteins that show an increased physical association with CRBN after IMiD treatment, we identified a novel IMiD substrate, Widely Interspaced Zinc Finger Motifs (WIZ). WIZ contains a C2H2 zinc finger domain, like several other substrates that were previously characterized. We demonstrate that IMiDs stabilize physical association of WIZ with CRBN, deplete WIZ steady state protein levels in a way that is dependent on E3 ligase activity, and enhance the rate of its degradation. Notably, proteins that assemble with WIZ are co-recruited to CRBN by IMiDs but are not degraded, illustrating the potential of targeted protein degradation to eliminate individual subunits of a protein complex. These findings suggest that systematic characterization of the full repertoire of proteins that are targeted for degradation by IMiD compounds will be required to better understand their biological effects

Single subunit degradation of WIZ, a lenalidomide- and pomalidomide dependent substrate of E3 ubiquitin ligase CRL4^(CRBN)

Immunomodulators (IMiDs) are an effective class of drugs used to treat blood cancers. IMiDs are believed to work by recruiting protein targets containing a β-hairpin motif for ubiquitination by E3 ubiquitin ligase complexes composed of cereblon (CRBN), Cullin-4a (CUL4a), DNA Damage Binding protein-1 (DDB1), and Ring Box-1 (RBX1). The ubiquitinated protein is subsequently degraded by the proteasome. By characterizing the repertoire of proteins that show an increased physical association with CRBN after IMiD treatment, we identified a novel IMiD substrate, Widely Interspaced Zinc Finger Motifs (WIZ). WIZ contains a C2H2 zinc finger domain, like several other substrates that were previously characterized. We demonstrate that IMiDs stabilize physical association of WIZ with CRBN, deplete WIZ steady state protein levels in a way that is dependent on E3 ligase activity, and enhance the rate of its degradation. Notably, proteins that assemble with WIZ are co-recruited to CRBN by IMiDs but are not degraded, illustrating the potential of targeted protein degradation to eliminate individual subunits of a protein complex. These findings suggest that systematic characterization of the full repertoire of proteins that are targeted for degradation by IMiD compounds will be required to better understand their biological effects

Complete Mitochondrial Genome Sequence of Three Tetrahymena Species Reveals Mutation Hot Spots and Accelerated Nonsynonymous Substitutions in Ymf Genes

The ciliate Tetrahymena, a model organism, contains divergent mitochondrial (Mt) genome with unusual properties, where half of its 44 genes still remain without a definitive function. These genes could be categorized into two major groups of KPC (known protein coding) and Ymf (genes without an identified function). To gain insights into the mechanisms underlying gene divergence and molecular evolution of Tetrahymena (T.) Mt genomes, we sequenced three Mt genomes of T.paravorax, T.pigmentosa, and T.malaccensis. These genomes were aligned and the analyses were carried out using several programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (ω) on individual codon sites and via a sliding window approach. Comparative genomic analysis indicated a conserved putative transcription control sequence, a GC box, in a region where presumably transcription and replication initiate. We also found distinct features in Mt genome of T.paravorax despite similar genome organization among these ∼47 kb long linear genomes. Another significant finding was the presence of at least one or more highly variable regions in Ymf genes where majority of substitutions were concentrated. These regions were mutation hotspots where elevated distances and the dn/ds ratios were primarily due to an increase in the number of nonsynonymous substitutions, suggesting relaxed selective constraint. However, in a few Ymf genes, accelerated rates of nonsynonymous substitutions may be due to positive selection. Similarly, on protein level the majority of amino acid replacements occurred in these regions. Ymf genes comprise half of the genes in Tetrahymena Mt genomes, so understanding why they have not been assigned definitive functions is an important aspect of molecular evolution. Importantly, nucleotide substitution types and rates suggest possible reasons for not being able to find homologues for Ymf genes. Additionally, comparative genomic analysis of complete Mt genomes is essential in identifying biologically significant motifs such as control regions

Molecular Evolution of Mitochondrial and Nuclear Genomes in Ciliate Terahymena

Ciliates are single cell eukaryotic organisms with two nuclei and unusual genome biology. The purpose of this project is to study the molecular evolution of macronuclear (MAC) and mitochondrial (Mt) genomes of ciliate protist Tetrahymena (T.) to decipher and interpret their evolutionary processes. Tetrahymena species contain rapidly evolving mitochondrial (Mt) genomes, which have apparently diverged significantly from the ancestral pattern leaving half of their 44 genes without an assigned function. Thus I sequenced three Mt genomes of Tetrahymena paravorax, Tetrahymena pigmentosa, and Tetrahymena malaccensis via shotgun sequencing. I found unique features in these linear 47kb long genomes including long telomeric repeats, long noncoding sequences, a pseudo-tRNA gene, unusual DNA secondary structure for T. paravorax, and the transcription initiation site, a GC box. More analyses were carried out using programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (oo) to discover mutation hotspots and relaxed selective constraint in Ymf genes. Hence I concluded that the presence of mutation hot spots in Tetrahymena Mt genomes is a major reason and obstacle in identifying their function.To study the MAC evolution I annotated a Tetrahymena thermophila chromosome, 1Mb in size, based on the translation of its possible open reading frames (ORFs) and their homologues in the GenBank. An objective of this study was to examine the TIGR automated annotation programs capability in identifying all of the biologically meaningful ORFs. Through searching the databases using BLAST I found proteins and ORFs with homologous domains that were not present in the TIGR automated annotation. Thus, I suggest phylogenetic footprinting to complement automated annotations. I also sought to reveal the extent of chromosome rearrangements between T. thermophila and the largest chromosome (LC) of Paramecium (P.) tetraurelia. Mapping all 463 putative proteins from the LC of P. tetraurelia to the chromosomes in T. thermophila genome revealed no orthologous chromosome. Interestingly, gene order between the Mt genome of these two ciliates was almost perfectly conserved, which suggests that the most plausible explanation for such an extensive gene order loss and rearrangement could be the developmentally programmed elimination of germ line sequences and randomization of MAC gene order

Molecular Evolution of Mitochondrial and Nuclear Genomes in Ciliate Terahymena

Ciliates are single cell eukaryotic organisms with two nuclei and unusual genome biology. The purpose of this project is to study the molecular evolution of macronuclear (MAC) and mitochondrial (Mt) genomes of ciliate protist Tetrahymena (T.) to decipher and interpret their evolutionary processes. Tetrahymena species contain rapidly evolving mitochondrial (Mt) genomes, which have apparently diverged significantly from the ancestral pattern leaving half of their 44 genes without an assigned function. Thus I sequenced three Mt genomes of Tetrahymena paravorax, Tetrahymena pigmentosa, and Tetrahymena malaccensis via shotgun sequencing. I found unique features in these linear 47kb long genomes including long telomeric repeats, long noncoding sequences, a pseudo-tRNA gene, unusual DNA secondary structure for T. paravorax, and the transcription initiation site, a GC box. More analyses were carried out using programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (oo) to discover mutation hotspots and relaxed selective constraint in Ymf genes. Hence I concluded that the presence of mutation hot spots in Tetrahymena Mt genomes is a major reason and obstacle in identifying their function.To study the MAC evolution I annotated a Tetrahymena thermophila chromosome, 1Mb in size, based on the translation of its possible open reading frames (ORFs) and their homologues in the GenBank. An objective of this study was to examine the TIGR automated annotation programs capability in identifying all of the biologically meaningful ORFs. Through searching the databases using BLAST I found proteins and ORFs with homologous domains that were not present in the TIGR automated annotation. Thus, I suggest phylogenetic footprinting to complement automated annotations. I also sought to reveal the extent of chromosome rearrangements between T. thermophila and the largest chromosome (LC) of Paramecium (P.) tetraurelia. Mapping all 463 putative proteins from the LC of P. tetraurelia to the chromosomes in T. thermophila genome revealed no orthologous chromosome. Interestingly, gene order between the Mt genome of these two ciliates was almost perfectly conserved, which suggests that the most plausible explanation for such an extensive gene order loss and rearrangement could be the developmentally programmed elimination of germ line sequences and randomization of MAC gene order