p21-activated protein kinase (PAK) 2 is a small GTPase-activated serine͞threonine kinase regulating various cytoskeletal functions and is cleaved by caspase-3 during apoptosis. We demonstrate that the caspase-cleaved PAK2 C-terminal kinase fragment (C-t-PAK2) is posttranslationally myristoylated, although myristoylation is typically a cotranslational process. Myristoylation and an adjacent polybasic domain of C-t-PAK2 are sufficient to redirect EGFP from the cytosol to membrane ruffles and internal membranes. Membrane localization and the ability of C-t-PAK2 to induce cell death are significantly reduced when myristoylation is abolished. In addition, the proper myristoylation-dependent membrane localization of C-t-PAK2 significantly increased signaling through the stress-activated c-Jun N-terminal kinase signaling pathway, which often regulates apoptosis. Interestingly, C-t-PAK2 promoted cell death without compromising mitochondrial integrity. Posttranslational myristoylation of caspase-cleaved proteins involved in cytoskeletal dynamics (e.g., PAK2, actin, and gelsolin) might be part of a unique series of mechanisms involved in the regulation of the later events of apoptosis.apoptosis ͉ cytoskeleton ͉ mitochondria ͉ membrane ͉ acylation

Fast, high-yielding, and selective bioorthogonal "click" reactions employing nontoxic reagents are in high demand for their great utility in the conjugation of biomolecules in live cells. Although a number of click reactions were developed for this purpose, many are associated with drawbacks and limitations that justify the development of alternative systems for both single- or dual-labeling applications. Recent reports have highlighted the potential of boronic ester formation as a bioorthogonal click reaction between abiotic boronic acids and diols. Boronic ester formation is a fast dehydrative process; however it is intrinsically reversible in aqueous medium. We designed and optimized a synergic system based on two bifunctional reagents, a thiosemicarbazide-functionalized nopoldiol and an ortho-acetyl arylboronic acid. Both reagents were shown to be chemically stable and nontoxic to HEK293T cells at concentrations as high as 50 μM. The resulting boronate/thiosemicarbazone adduct is a medium-sized ring that forms rapidly and irreversibly without any catalyst at low μM concentrations, in neutral buffer, with a rate constant of 9 M s as measured by NMR spectroscopy. Control experiments in the presence of competing boronic acids showed no crossover side-products and confirmed the stability and lack of reversibility of the boronate/thiosemicarbazone conjugates. Formation of the conjugates is not affected by the presence of biological diols such as fructose, glucose, and catechol, and the thiosemicarbazide-functionalized nopoldiol is inert to aldehyde electrophiles of the sort found on protein-bound glyoxylyl units. The suitability of this system in the cell-surface labeling of live cells was demonstrated using a SNAP-tag approach to install the boronic acid reagent onto the extracellular domain of the Beta-2 adrenergic receptor in HEK293T cells, followed by incubation with the optimal thiosemicarbazide-functionalized nopoldiol reagent labeled with fluorescein dye. Successful visualization by fluorescence microscopy was possible with a reagent concentration as low as 10 μM, thus confirming the potential of this system in biological applications.

PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strain pex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels in pex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane. INTRODUCTIONPeroxisomes, together with the glyoxysomes of plants and the glycosomes of trypanosomes, make up the microbody family of organelles (de Duve, 1996). In electron micrographs, peroxisomes appear spherical in shape, surrounded by a single unit membrane and containing a granular matrix and sometimes a paracrystalline core. The functions of peroxisomes vary depending on the organism, cell type, and physiological conditions. Functions that have been conserved from yeast to humans include the ␤-oxidation of fatty acids and the decomposition of hydrogen peroxide by catalase (for reviews, see Lazarow and Fujiki, 1985;van den Bosch et al., 1992). Proteins that are required for the proper functioning and biogenesis of peroxisomes have collectively been termed peroxins .The importance of peroxisomes for human growth and development is underscored by a group of genetic disorders known as the peroxisome biogenesis disorders (PBD), including Zellweger syndrome, rhizomelic chondrodysplasia punctata, and neonatal adrenoleukodystrophy, in which peroxisomes fail to assemble normally (for reviews, see Lazarow and Moser, 1994;Fujiki, 1997Fujiki, , 2000. A great deal of attention has been paid recently to defining the molecular bases of these diseases, in particular...

An innovative application of metal−organic frameworks (MOFs) is in biomedical materials. To treat bone demineralization, which is a hallmark of osteoporosis, biocompatible MOFs (bioMOFs) have been proposed in which various components, such as alkaline-earth cations and bisphosphonate molecules, can be delivered to maintain normal bone density. Multicomponent bioMOFs that release several components simultaneously at a controlled rate thus offer an attractive solution. We report two new bioMOFs, comprising strontium and calcium ions linked by p-xylylenebisphosphonate molecules that release these three components and display no cytotoxic effects on human osteosarcoma cells. Varying the Sr 2+ /Ca 2+ ratio in these bioMOFs causes the rate of ions dissolving into simulated body fluid to be unique; along with the ability to adsorb proteins, this property is crucial for future efforts in drugrelease control and promotion of mineral formation. The one-pot synthesis of these bioMOFs demonstrates the utility of MOF design strategies.

Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11-dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non-reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis, and homology modeling of these enzymes has identified variable surfaces and conserved motifs that are implicated in product formation.