Domain unfolding in neurofilament sidearms: effects of phosphorylation and ATP

Lateral projections of neurofilaments (NF) called sidearms (SA) affect axon stability and caliber. SA phosphorylation is thought to modulate inter-NF distance and interactions between NF and other subcellular organelles. SA were probed by atomic force microscopy (AFM) and dynamic light scattering (DLS) as a function of phosphorylation and ATP content. DLS shows SA are larger when phosphorylated, and AFM shows four unfoldable domains in SA regardless of phosphorylation state or the presence of ATP. However, the native phosphorylated SA requires three-fold higher force to unfold by AFM than dephosphorylated SA, suggesting a less pliant as well as larger structure when phosphorylated.     

Further characterization of the putative human isopeptidase T catalytic site

The human isopeptidase T (isoT) is a zinc-binding deubiquitinating enzyme involved in the disassembly of free K48-linked polyubiquitin chains into ubiquitin monomers. The catalytic site of this enzyme is thought to be composed of Cys335, Asp435, His786 and His795. These four residues were site-directed mutagenized. None of the mutants were able to cleave a peptide-linked ubiquitin dimer. Similarly, C335S, D435N and H795N mutants had virtually no activity against a K48-linked isopeptide ubiquitin dimer, which is an isoT-specific substrate that mimics the K48-linked polyubiquitin chains. On the other hand, the H786N mutant retained a partial activity toward the K48-linked substrate, suggesting that the His786 residue might not be part of the catalytic site. None of the mutations significantly affected the capacity of isoT to bind ubiquitin and zinc. Thus, the catalytic site of UBPs could resemble that of other cysteine proteases, which contain one Cys, one Asp and one His.     

Genetic control of extracellular protease synthesis in the yeast Yarrowia lipolytica

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.     

Non-antioxidant molecular functions of alpha-tocopherol (vitamin E)

alpha-Tocopherol (the major vitamin E component) regulates key cellular events by mechanisms unrelated with its antioxidant function. Inhibition of protein kinase C (PKC) activity and vascular smooth muscle cell growth by alpha-tocopherol was first described by our group. Later, alpha-tocopherol was shown to inhibit PKC in various cell types with consequent inhibition of aggregation in platelets, of nitric oxide production in endothelial cells and of superoxide production in neutrophils and macrophages. alpha-Tocopherol diminishes adhesion molecule, collagenase and scavenger receptor (SR-A and CD36) expression and increases connective tissue growth factor expression.     

Fibroblast growth factor 18 influences proximal programming during lung morphogenesis

The structure and functions of the airways of the lung change dramatically along their lengths. Large-diameter conducting airways are supported by cartilaginous rings and smooth muscle tissue and are lined by ciliated and secretory epithelial cells that are involved in mucociliary clearance. Smaller peripheral airways formed during branching morphogenesis are lined by cuboidal and squamous cells that facilitate gas exchange to a network of fine capillaries. The factors that mediate formation of these changing cell types and structures along the length of the airways are unknown. We report here that conditional expression of fibroblast growth factor (FGF)-18 in epithelial cells of the developing lung caused the airway to adopt structural features of proximal airways. Peripheral lung tubules were markedly diminished in numbers, whereas the size and extent of conducting airways were increased. Abnormal smooth muscle and cartilage were found in the walls of expanded distal airways, which were accompanied by atypically large pulmonary blood vessels. Expression of proteins normally expressed in peripheral lung tubules, including SP-B and pro-SP-C, was inhibited. FGF-18 mRNA was detected in normal mouse lung in stromal cells surrounding proximal airway cartilage and in peripheral lung mesenchyme. Effects were unique to FGF-18 because expression of other members of the FGF family had different consequences. These data show that FGF-18 is capable of enhancing proximal and inhibiting peripheral programs during lung morphogenesis.     

Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.     

Late endosome motility depends on lipids via the small GTPase Rab7

We report that lipids contribute to regulate the bidirectional motility of late endocytic compartments. Late endocytic vesicles loaded with cholesterol lose their dynamic properties, and become essentially immobile, including in cells from Niemann-Pick C patients. These vesicles then retain cytoplasmic dynein activity, but seem to be unable to acquire kinesin activity, eventually leading to paralysis. Our data suggest that this defect depends on the small GTPase Rab7, since the motility of vesicles loaded with cholesterol can be restored by the Rab7 inhibitory mutant N125I. Conversely, wild-type Rab7 overexpression mimics the effects of cholesterol on motility in control cells. Consistently, cholesterol accumulation increases the amounts of membrane-associated Rab7, and inhibits Rab7 membrane extraction by the guanine nucleotide dissociation inhibitor. Our observations thus indicate that cholesterol contributes to regulate the Rab7 cycle, and that Rab7 in turn controls the net movement of late endocytic elements. We conclude that motor functions can be regulated by the membrane lipid composition via the Rab7 cycle.