TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.

Polyglutamylation is a dynamic posttranslational modification where glutamate residues are added to substrate proteins by 8 tubulin tyrosine ligase-like (TTLL) family members (writers) and removed by the 6 member Nna1/CCP family of carboxypeptidases (erasers). Genetic disruption of polyglutamylation leading to hyperglutamylation causes neurodegenerative phenotypes in humans and animal models; the best characterized being the Purkinje cell degeneration (pcd) mouse, a mutant of the gene encoding Nna1/CCP1, the prototypic eraser. Emphasizing the functional importance of the balance between glutamate addition and elimination, loss of TTLL1 prevents Purkinje cell degeneration in pcd. However, whether Ttll1 loss protects other vulnerable neurons in pcd, or if elimination of other TTLLs provides protection is largely unknown. Here using a mouse genetic rescue strategy, we characterized the contribution of Ttll1, 4, 5, 7, or 11 to the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mutants. Ttll1 deficiency attenuates Purkinje cell loss and function and reduces olfactory bulb mitral cell death and retinal photoreceptor degeneration. Moreover, degeneration of photoreceptors in pcd is preceded by impaired rhodopsin trafficking to the rod outer segment and likely represents the causal defect leading to degeneration as this too is rescued by elimination of TTLL1. Although TTLLs have similar catalytic properties on model substrates and several are highly expressed in Purkinje cells (e.g. TTLL5 and 7), besides TTLL1 only TTLL4 deficiency attenuated degeneration of Purkinje and mitral cells in pcd. Additionally, TTLL4 loss partially rescued photoreceptor degeneration and impaired rhodopsin trafficking. Despite their common properties, the polyglutamylation profile changes promoted by TTLL1 and TTLL4 deficiencies in pcd mice are very different. We also report that loss of anabolic TTLL5 synergizes with loss of catabolic Nna1/CCP1 to promote photoreceptor degeneration. Finally, male infertility in pcd is not rescued by loss of any Ttll. These data provide insight into the complexity of polyglutamate homeostasis and function in vivo and potential routes to ameliorate disorders caused by disrupted polyglutamylation.

Glycosylation of Cblns attenuates their receptor binding.

Cbln1 is the prototype of a family (Cbln1-Cbln4) of secreted glycoproteins and is essential for normal synapse structure and function in cerebellum by bridging presynaptic Nrxn to postsynaptic Grid2. Here we report the effects of glycosylation on the in vitro receptor binding properties of Cblns. Cbln1, 2 and 4 harbor two N-linked glycosylation sites, one at the N-terminus is in a region implicated in Nrxn binding and the second is in the C1q domain, a region involved in Grid2 binding. Mutation (asparagine to glutamine) of the N-terminal site, increased neurexin binding whereas mutation of the C1q site markedly increased Grid2 binding. These mutations did not influence subunit composition of Cbln trimeric complexes (mediated through the C1q domain) nor their assembly into hexamers (mediated by the N-terminal region). Therefore, glycosylation likely masks the receptor binding interfaces of Cblns. As Cbln4 has undetectable Grid2 binding in vitro we assessed whether transgenic expression of wild type Cbln4 or its glycosylation mutants rescued the Cbln1-null phenotype in vivo. Cbln4 partially rescued and both glycosylation mutants completely rescued ataxia in cbln1-null mice. Thus Cbln4 has intrinsic Grid2 binding that is attenuated by glycosylation, and glycosylation mutants exhibit gain of function in vivo.

Synthetic genetic array screen identifies PP2A as a therapeutic target in Mad2-overexpressing tumors.

The spindle checkpoint is essential to ensure proper chromosome segregation and thereby maintain genomic stability. Mitotic arrest deficiency 2 (Mad2), a critical component of the spindle checkpoint, is overexpressed in many cancer cells. Thus, we hypothesized that Mad2 overexpression could specifically make cancer cells susceptible to death by inducing a synthetic dosage lethality defect. Because the spindle checkpoint pathway is highly conserved between yeast and humans, we performed a synthetic genetic array analysis in yeast, which revealed that Mad2 overexpression induced lethality in 13 gene deletions. Among the human homologs of candidate genes, knockdown of PPP2R1A, a gene encoding a constant regulatory subunit of protein phosphatase 2, significantly inhibited the growth of Mad2-overexpressing tumor cells. PPP2R1A inhibition induced Mad2 phosphorylation and suppressed Mad2 protein levels. Depletion of PPP2R1A inhibited colony formation of Mad2-overexpressing HeLa cells but not of unphosphorylated Mad2 mutant-overexpressing cells, suggesting that the lethality induced by PP2A depletion in Mad2-overexpressing cells is dependent on Mad2 phosphorylation. Also, the PP2A inhibitor cantharidin induced Mad2 phosphorylation and inhibited the growth of Mad2-overexpressing cancer cells. Aurora B knockdown inhibited Mad2 phosphorylation in mitosis, resulting in the blocking of PPP2R1A inhibition-induced cell death. Taken together, our results strongly suggest that PP2A is a good therapeutic target in Mad2-overexpressing tumors.

The Cbln family of proteins interact with multiple signaling pathways.

Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic ß-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.

Sgt1 dimerization is negatively regulated by protein kinase CK2-mediated phosphorylation at Ser361.

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser(361) on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.

Sgt1 dimerization is required for yeast kinetochore assembly.

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1-211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.

SCF E3-mediated autoubiquitination negatively regulates activity of Cdc34 E2 but plays a nonessential role in the catalytic cycle in vitro and in vivo.

One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii.

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.

Sgt1 associates with Hsp90: an initial step of assembly of the core kinetochore complex.

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1, together with Skp1, is required for assembly of the core kinetochore complex (CBF3) via Ctf13 activation. Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone. We have found that Sgt1 interacts with Hsp90 in yeast. We also have determined that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide repeat motifs. Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1. We found that Hsp90's binding to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13, the F-box core kinetochore protein. Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.

Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint.

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.