Aggregation of expanded polyglutamine domain in yeast leads to defects in endocytosis

The role of aggregation of abnormal proteins in cellular toxicity is of general importance for understanding many neurological disorders. Here, using a yeast model, we demonstrate that mutations in many proteins involved in endocytosis and actin function dramatically enhance the toxic effect of polypeptides with an expanded polyglutamine (polyQ) domain. This enhanced cytotoxicity required polyQ aggregation and was dependent on the yeast protein Rnq1 in its prion form. In wild-type cells, expression of expanded polyQ followed by its aggregation led to specific and acute inhibition of endocytosis, which preceded growth inhibition. Some components of the endocytic machinery were efficiently recruited into the polyQ aggregates. Furthermore, in cells with polyQ aggregates, cortical actin patches were delocalized and actin was recruited into the polyQ aggregates. Aggregation of polyQ in mammalian HEK293 cells also led to defects in endocytosis. Therefore, it appears that inhibition of endocytosis is a direct consequence of polyQ aggregation and could significantly contribute to cytotoxicity.     

On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.     

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.     

Impaired DNA replication within progenitor cell pools promotes leukemogenesis

Impaired cell cycle progression can be paradoxically associated with increased rates of malignancies. Using retroviral transduction of bone marrow progenitors followed by transplantation into mice, we demonstrate that inhibition of hematopoietic progenitor cell proliferation impairs competition, promoting the expansion of progenitors that acquire oncogenic mutations which restore cell cycle progression. Conditions that impair DNA replication dramatically enhance the proliferative advantage provided by the expression of Bcr-Abl or mutant p53, which provide no apparent competitive advantage under conditions of healthy replication. Furthermore, for the Bcr-Abl oncogene the competitive advantage in contexts of impaired DNA replication dramatically increases leukemogenesis. Impaired replication within hematopoietic progenitor cell pools can select for oncogenic events and thereby promote leukemia, demonstrating the importance of replicative competence in the prevention of tumorigenesis. The demonstration that replication-impaired, poorly competitive progenitor cell pools can promote tumorigenesis provides a new rationale for links between tumorigenesis and common human conditions of impaired DNA replication such as dietary folate deficiency, chemotherapeutics targeting dNTP synthesis, and polymorphisms in genes important for DNA metabolism.     

Synthesis and DNA binding properties of dioxime-peptide nucleic acids

Peptide nucleic acids (PNAs) C- or N-modified with dioxime ligands were prepared by solid-phase synthesis using iron(II)-clathrochelates as protected dioxime building blocks. These PNA bind complementary DNA sequence specifically, though with much reduced affinity in comparison with nonmodified PNA. The dioxime-PNA conjugates bind Cu2+ and Ni2+ at microM concentration.     

It works: combinatorial biosynthesis for generating novel glycosylated compounds

Combinatorial biosynthesis is a valuable method to generate novel glycosylated natural products. By coexpression of deoxysugar gene cassettes and genes from the staurosporine biosynthetic gene cluster it has now been applied to the generation of novel staurosporine derivatives. The work of Salas and co-workers is highlighted in this article.     

Hybridization dependent cleavage of internally modified disulfide-peptide nucleic acids

Selectivity of the cleavage of single stranded over hybridized forms of internally modified disulfide-peptide nucleic acids (PNA) has been optimized using a series of phosphines and thiols, which have different sizes and charges. For the most selective cleaver found (tris-(carboxyethyl)-phosphine), reactivity of single stranded PNA is 33 times higher than that of the PNA-DNA duplex. Selectivity of single stranded disulfide-PNA cleavage has been explained in terms of electrostatic interaction between the substrate and the cleaver.