Mechanism of folding and activation of subtilisin kexin isozyme-1(SKI-1)/site-1 protease (S1P) [Protein Structure and Folding]

December 8th, 2015 by

The proprotein convertase subtilisin kexin isozyme-1(SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multi-step process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure, but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition possibly allowing more subtle fine-tuning of the maturation process.

Acetoacetate accelerates muscle regeneration and ameliorates muscular dystrophy in mice [Cell Biology]

December 8th, 2015 by

Acetoacetate (AA) is a ketone body and acts as a fuel to supply energy for cellular activity of various tissues. Here, we uncovered a novel function of AA in promoting muscle cell proliferation. Notably, the functional role of AA in regulating muscle cell function is further evidenced by its capability to accelerates muscle regeneration in normal mice and ameliorates muscular dystrophy in mdx mice. Mechanistically, our data from multi-parameter analyses consistently support the notion that AA plays a non-metabolic role in regulating muscle cell function. Finally, we show that AA exerts its function through activation of the Mek1-Erk1/2-cyclin D1 pathway, revealing a novel mechanism in which AA serves as a signaling metabolite in mediating muscle cell function. Our findings highlight profound functions of a small metabolite as signaling molecule in mammalian cells.

Regulation of the Water Channel Aquaporin-2 via 14-3-3 Theta ({theta}) and Zeta ({zeta}) [Membrane Biology]

December 8th, 2015 by

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of sigma (σ), all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3 beta (β) and zeta (ζ), whereas levels of 14-3-3 eta (η) and theta (θ) were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2:14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3 ζ and θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2:14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl-terminus, with ser256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3 ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life and reduced AQP2 levels. In contrast, knockdown of 14-3-3 θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3 θ and ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation and degradation.

Trypanosome Lytic Factor-1 Initiates Oxidation-Stimulated Osmotic Lysis of Trypanosoma brucei brucei [Membrane Biology]

December 8th, 2015 by Styer, A. L., Hajduk, S. L.

Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. b. gambiense and T. b. rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate specific apolipoproteins: the ion-channel forming toxin apolipoproteinL1 (ApoL1), and the hemoglobin (Hb) scavenger haptoglobin related protein (Hpr). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. b. brucei. The lipophilic antioxidant DPPD protected TLF-1 treated T. b. brucei from lysis. Conversely, lysis of TLF-1 treated T. b. brucei was increased by addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the Fe-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knockout of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1 treated T. b. brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1 treated T. b. brucei became rapidly susceptible to hypotonic lysis. T. b. brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane which sensitizes T. b. brucei to oxidation-stimulated osmotic lysis.

Antioxidant defenses of Francisella tularensis modulate macrophage function and production of proinflammatory cytokines [Microbiology]

December 7th, 2015 by

Francisella tularensis, the causative agent of a fatal human disease known as tularemia has been used in the bioweapon programs of several countries in the past, and now is considered a potential bioterror agent. Extreme infectivity and virulence of F. tularensis is due to its ability to evade immune detection and to suppress hosts innate immune responses. However, Francisella encoded factors and mechanisms responsible for causing immune suppression are not completely understood. Macrophages and neutrophils generate Reactive Oxygen-Nitrogen Species (ROS,RNS) as a defense mechanism for the clearance of phagocytosed microorganisms. ROS serve a dual role; at high concentrations they act as microbicidal effector molecules that destroy intracellular pathogens and at low concentrations, they serve as secondary signaling messengers that regulate the expression of various inflammatory mediators. We hypothesized that antioxidant defenses of F. tularensis maintain redox-homeostasis in infected macrophages to prevent activation of redox-sensitive signaling components which ultimately result in suppression of pro-inflammatory cytokine production and macrophage microbicidal activity. We demonstrate that antioxidant enzymes of F. tularensis prevent the activation of redox-sensitive MAPK signaling components, NF-kappaB signaling and the production of pro-inflammatory cytokines by inhibiting the accumulation of ROS in infected macrophages. We also report that F. tularensis inhibits ROS-dependent autophagy to promote its intramacrophage survival. Collectively, this study reveals novel pathogenic mechanisms adopted by F. tularensis to modulate macrophage innate immune functions to create an environment permissive for its intracellular survival and growth.

Information Transfer in Gonadotropin-Releasing Hormone (GnRH) Signaling: Extracellular Signal-Regulated Kinase (ERK)-Mediated Feedback Loops Control Hormone Sensing. [Computational Biology]

December 7th, 2015 by

Cell signaling pathways are noisy communication channels and statistical measures derived from information theory can be used to quantify the information they transfer. Here we use single cell signaling measures to calculate mutual information (MI) as a measure of information transfer via gonadotropin-releasing hormone receptors (GnRHR) to extracellular signal-regulated kinase (ERK) or nuclear factor of activated T-cells (NFAT). This revealed MI values <1 Bit, implying that individual GnRH-responsive cells cannot unambiguously differentiate even two equally probable input concentrations. Addressing possible mechanisms for mitigation of information loss we focused on the ERK pathway and developed a stochastic activation model incorporating negative feedback and constitutive activity. Model simulations revealed interplay between fast (min) and slow (min-hr) negative feedback loops with maximal information transfer at intermediate feedback levels. Consistent with this, experiments revealed that reducing negative feedback (by expressing catalytically inactive ERK2) and increasing negative feedback (by Egr1-driven expression of dual-specificity phosphatase 5 (DUSP5)) both reduced information transfer from GnRHR to ERK. It was also reduced by blocking protein synthesis (in order to prevent GnRH from increasing DUSP expression) but did not differ for different GnRHR that do or do not undergo rapid homologous desensitization. Thus, the first statistical measures of information transfer via these receptors reveals that individual cells are unreliable sensors of GnRH concentration and that this reliability is maximal at intermediate levels of ERK-mediated negative feedback but is not influenced by receptor desensitization.
  • Posted in Journal of Biological Chemistry, Publications
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Selenoprotein expression in macrophages is critical for optimal clearance of parasitic helminth Nippostrongylus brasiliensis [Lipids]

December 7th, 2015 by

The plasticity of macrophages is evident in helminthic parasite infections, providing protection from inflammation. Previously we demonstrated that the micronutrient selenium (Se) induces a phenotypic switch in macrophage activation from a classically activated (pro-inflammatory; M1/CAM) towards an alternatively activated (anti-inflammatory; M2/AAM) phenotype, where cyclooxygenase (COX)-dependent cyclopentenone prostaglandin J2 (15d-PGJ2) plays a key role. Here, we hypothesize that dietary Se modulates macrophage polarization towards an AAM phenotype to assist in the increasing clearance of adult Nippostrongylus brasiliensis, a gastrointestinal nematode parasite. Mice on a Se adequate (0.08 ppm) diet significantly augmented intestinal AAM presence, while decreasing adult worms and fecal egg production when compared to infection of mice on Se deficient (<0.01 ppm) diet. Further increase in dietary Se to supraphysiological levels (0.4 ppm) had very little or no impact on worm expulsion. Normal adult worm clearance and enhanced AAM marker expression were observed in the Se supplemented Trspfl/flCreWT mice that express selenoproteins driven by tRNASec (Trsp), while N. brasiliensis infected Trspfl/flCreLysM Se supplemented mice showed a decreased clearance, with lowered intestinal expression of several AAM markers. Inhibition of the COX pathway with indomethacin resulted in delayed worm expulsion in Se adequate mice. This was rescued with 15d-PGJ2, which partially recapitulated the effect of Se supplementation on fecal egg output in addition to increasing markers of AAMs in the small intestine. Antagonism of PPARg blocked the effect of Se. These results suggest that optimal expression of selenoproteins and Se-dependent production of COX-derived endogenous prostanoids, such as 15d-PGJ2, may regulate AAM activation to enhance anti-helminthic parasite responses.

{alpha}-Synuclein Amyloid Fibrils with Two Entwined, Asymmetrically Associated, Protofibrils [Protein Structure and Folding]

December 7th, 2015 by

Parkinson's disease (PD) and other progressive neurodegenerative conditions are characterized by the intracerebral presence of Lewy Bodies, containing amyloid fibrils of α-Synuclein (αS). We have used cryo-electron microscopy and scanning transmission electron microscopy to study in vitro-assembled fibrils. These fibrils are highly polymorphic. Focusing on twisting fibrils with an inter-crossover spacing of 77 nm, our reconstructions showed them to consist of paired protofibrils. STEM mass-per-length data gave one subunit per 0.47 nm axial rise per protofibril, consistent with a superpleated β-structure. The STEM images show two thread-like densities running along each of these fibrils, which we interpret as ladders of metal ions. These threads confirmed the two-protofibril architecture of the 77 nm twisting fibrils and allowed us to identify this morphotype in STEM micrographs. Some other - but not all - fibril morphotypes also exhibit dense threads, implying that they also present a putative metal binding site. We propose a molecular model for the protofibril and suggest that polymorphic variant fibrils have different numbers of protofibrils, differently associated.

Evidence for conservation of the calcitonin superfamily and activity-regulating mechanisms in the basal chordate Branchiostoma floridae: insight into the molecular and functional evolution in chordates [Cell Biology]

December 7th, 2015 by

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell-surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R-Bf-RAMP-LP complexes resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenmedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.
  • Posted in Journal of Biological Chemistry, Publications
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Antigenic determinants of the bilobal cockroach allergen Bla g 2 [Immunology]

December 7th, 2015 by

Bla g 2 is a major indoor cockroach allergen associated with the development of asthma. Antigenic determinants on Bla g 2 were analyzed by mutagenesis based on the structure of the allergen alone and in complex with monoclonal antibodies that interfere with IgE antibody binding. The structural analysis revealed mechanisms of allergen-antibody recognition through cation-π interactions. Single and multiple Bla g 2 mutants were expressed in Pichia pastoris and purified. The triple mutant K132A-K251A-F162Y showed a ~100 reduced capacity to bind IgE, while preserving the native molecular fold, as proven by X-ray crystallography. This mutant was still able to induce mast cell release. T-cell responses were assessed by analyzing Th1/Th2 cytokine production and CD4+ T-cell phenotype in PBMC cultures. Whereas T-cell activating capacity was similar for the KKF mutant and Bla g 2 based on CD25 expression, the KKF mutant was a weaker inducer of the Th2 cytokine IL-13. Furthermore, this mutant induced IL-10 from a non-T-cell source at higher levels that those induced by Bla g 2. Our findings demonstrate that a rational design of site-directed mutagenesis was effective in producing a mutant with only 3 amino acid substitutions that maintained the same fold as wild type Bla g 2. These residues, which were involved in IgE antibody binding, endowed Bla g 2 with a T cell modulatory capacity. The antigenic analysis of Bla g 2 will be useful for the subsequent development of recombinant allergen vaccines.