The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N‐terminus

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.     

Expression of l-3-phosphoserine phosphatase is regulated by reconstituted basement membrane

Reconstituted basement membrane (Matrigel) promotes differentiation of endometrial adenocarcinoma cells in vitro. However, little is known about the molecular basis of these in vitro differentiation processes. Using differential display RT-PCR to search for potential molecular markers we screened for genes which respond to contact to basement membrane by alteration of expression levels. Here we report that the cDNA MT32 represents an mRNA with a time dependent biphasic response pattern to contact to basement membrane. Characterizing MT32 revealed that the sequence of MT32 is identical to l-3-phosphoserine phosphatase. PCR analysis of l-3-phosphoserine phosphatase expression surprisingly revealed at least three variants of this enzyme. In summary, and in view of the literature, l-3-phosphoserine phosphatase and potential variants or family members represent molecular markers to study regulation of gene expression by components of the extracellular matrix. In conclusion, l-3-phosphoserine phosphatase(s) may be important in endometrial carcinogenesis since this enzyme synthesizes important metabolic intermediates which serve both as building blocks for peptide synthesis and for signal transducing molecules.     

Adenovirus-mediated expression of caveolin-1 in mouse liver increases plasma high-density lipoprotein levels

Caveolae are 50-100 nm plasma membrane invaginations, which function in cell signaling and transcytosis, as well as in regulating cellular cholesterol homeostasis. These subcompartments of the plasma membrane are characterized by the presence of caveolin proteins. Recent studies have indicated that caveolae may be involved in the regulation of cellular cholesterol efflux to HDL, as well as selective uptake mediated by SR-BI. In the present study, we have determined the effect of caveolin-1 overexpression in mouse liver on plasma lipoprotein metabolism. We evaluated this effect using an adenovirus-mediated gene delivery system. C57BL/6J mice were injected with adenoviruses encoding either caveolin-1 (Adcav-1) or green fluorescent protein (AdGFP) together with a transactivator adenovirus (AdtTA). We found that, after adenovirus injection, caveolin-1 was overexpressed in hepatocytes. Moreover, the recombinant protein was localized to the plasma membrane. We also found that caveolin-1 overexpression induced a marked change in the lipoprotein profile of injected animals. In caveolin-1 overexpressing animals, plasma HDL-cholesterol levels were found to be approximately 2-fold elevated, as compared with control animals. To determine the effect of caveolin-1 on SR-BI-mediated selective uptake, we infected murine hepatocytes in culture with an adenoviral vector carrying the caveolin-1 cDNA or GFP as a control protein. We show that, in primary cultures of hepatocytes, caveolin-1 inhibits DiI-HDL uptake mediated by SR-BI. This result would mechanistically explain the increased plasma HDL-cholesterol levels we observed in caveolin-1 adenovirus-injected animals. In addition, caveolin-1 expression increased the secretion of apolipoprotein A-I in cultured hepatocytes and increased apolipoprotein A-I plasma levels in mice. Our study therefore demonstrates an important role for caveolin-1 in regulating HDL metabolism.     

Activation of MAPKs by angiotensin II in vascular smooth muscle cells. Metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAPK but not for JNK

In cultured vascular smooth muscle cells (VSMC), the vasculotrophic factor, angiotensin II (AngII) activates three major MAPKs via the G(q)-coupled AT1 receptor. Extracellular signal-regulated kinase (ERK) activation by AngII requires Ca(2+)-dependent "transactivation" of the EGF receptor that may involve a metalloprotease to stimulate processing of an EGF receptor ligand from its precursor. Whether EGF receptor transactivation also contributes to activation of other members of MAPKs such as p38MAPK and c-Jun N-terminal kinase (JNK) by AngII remains unclear. In the present study, we have examined the effects of a synthetic metalloprotease inhibitor BB2116, and the EGF receptor kinase inhibitor AG1478 on AngII-induced activation of MAPKs in cultured VSMC. BB2116 markedly inhibited ERK activation induced by AngII or the Ca(2+) ionophore without affecting the activation by EGF or PDGF. BB2116 as well as HB-EGF neutralizing antibody inhibited the EGF receptor transactivation by AngII, suggesting a critical role of HB-EGF in the metalloprotease-dependent EGF receptor transactivation. In addition to the ERK activation, activation of p38MAPK and JNK by AngII was inhibited by an AT1 receptor antagonist, RNH6270. and EGF markedly activate p38MAPK, whereas but not EGF markedly activates JNK, indicating the possible contribution of the EGF receptor transactivation to the p38MAPK activation. The findings that both BB2116 and AG1478 specifically inhibited activation of p38MAPK but not JNK by AngII support this hypothesis. From these data, we conclude that ERK and p38MAPK activation by AngII requires the metalloprotease-dependent EGF receptor transactivation, whereas the JNK activation is regulated without involvement of EGF receptor transactivation.     

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.     

Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.     

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.     

Spectroscopic studies of the low-lying singlet excited electronic states and photochemical properties of carotenoids

Investigations of the singlet excited state properties of carotenoids using steady-state fluorescence, transient absorption pump-probe, two-photon excitation, and resonance Raman excitation spectroscopies are described. The application of these experimental techniques to the specific problem of determining the S1 excited energies of carotenoids is discussed in detail, and the recent literature pertaining to the assignment of charge transfer states in carotenoids and states described as having particular pseudoparity elements is reviewed. Hypothetical schemes for how these states may account for some of the dynamic and photochemical behavior of carotenoids are presented.     

Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-alpha (TNF-alpha). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a "sheddase" during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-alpha or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development.     

Isolation of a new Thermoanaerobacterium thermosaccharolyticum strain (FH1) producing a thermostable dextranase

A Gram-positive spore-forming thermophilic strict anaerobic bacterium, designated FH1, was isolated from enrichments at 65 degrees C with dextran as sole carbon and energy source. A sequence analysis of the 16S rRNA gene revealed 99.2% identity of FH1 to Thermoanaerobacterium thermosaccharolyticum. Furthermore, the substrate spectra of both organisms were similar. It was therefore concluded that FH1 represents a new strain within the species T. thermosaccharolyticum. The optimal growth temperature of strain FH1 was 68 degrees C. The isolated organism produced a thermostable and thermoactive dextranase with a native molecular mass of approximately 200,000 Da. The enzyme was concentrated from the cell-free culture supernatant by ammonium sulfate precipitation. The resulting crude dextranase exhibited optimal activity from 65 to 70 degrees C and a pH optimum of 5.5.