An Approach for Reducing Unwanted Oligomerisation of DsRed Fusion Proteins

Biochemical and Biophysical Research Communications. Nov, 2002  |  Pubmed ID: 12419311

Oligomerisation of the red fluorescent protein, DsRed, can interfere with the localisation and function of proteins to which it is fused. We demonstrate an approach that may help to reduce significantly the impact of oligomerisation on the biology of the protein fusion partner. Growth of yeast (Saccharomyces cerevisiae) cells expressing ATP synthase containing subunit gamma-DsRed fusion was compromised relative to control cells. Furthermore, ATP synthase was found to exist as oligomeric structures when isolated under conditions where monomers would normally be present. The compromised growth phenotype was partially reversed and the oligomerisation of the ATP synthase reduced when a non-fluorescent variant of DsRed not fused to another protein was targeted to the mitochondrion in addition to the gamma-DsRed fusion protein. This strategy may also be applicable to the reduction of unwanted interactions between fusion proteins that contain the normally dimeric fluorescent proteins HcRed or Renilla GFP.

Identification of a Novel Domain in Two Mammalian Inositol-polyphosphate 5-phosphatases That Mediates Membrane Ruffle Localization. The Inositol 5-phosphatase Skip Localizes to the Endoplasmic Reticulum and Translocates to Membrane Ruffles Following Epidermal Growth Factor Stimulation

The Journal of Biological Chemistry. Mar, 2003  |  Pubmed ID: 12536145

SKIP (skeletal muscle and kidney enriched inositol phosphatase) is a recently identified phosphatidylinositol 3,4,5-trisphosphate- and phosphatidylinositol 4,5-bisphosphate-specific 5-phosphatase. In this study, we investigated the intracellular localization of SKIP. Indirect immunofluorescence and subcellular fractionation showed that, in serum-starved cells, both endogenous and recombinant SKIP colocalized with markers of the endoplasmic reticulum (ER). Following epidermal growth factor (EGF) stimulation, SKIP transiently translocated to plasma membrane ruffles and colocalized with submembranous actin. Data base searching demonstrated a novel 128-amino acid domain in the C terminus of SKIP, designated SKICH for SKIP carboxyl homology, which is also found in the 107-kDa 5-phosphatase PIPP and in members of the TRAF6-binding protein family. Recombinant SKIP lacking the SKICH domain localized to the ER, but did not translocate to membrane ruffles following EGF stimulation. The SKIP SKICH domain showed perinuclear localization and mediated EGF-stimulated plasma membrane ruffle localization. The SKICH domain of the 5-phosphatase PIPP also mediated plasma membrane ruffle localization. Mutational analysis identified the core sequence within the SKICH domain that mediated constitutive membrane association and C-terminal sequences unique to SKIP that contributed to ER localization. Collectively, these studies demonstrate a novel membrane-targeting domain that serves to recruit SKIP and PIPP to membrane ruffles.

The Production, Purification and Crystallization of a Pocilloporin Pigment from a Reef-forming Coral

Acta Crystallographica. Section D, Biological Crystallography. Mar, 2003  |  Pubmed ID: 12595737

Reef-building corals contain fluorescent pigments termed pocilloporins that function by regulating the light environment of coral and acting as a photoprotectant in excessive sunlight. These pocilloporins are related to the monomeric green fluorescent protein and the tetrameric DsRed fluorescent proteins, which have widespread use as biotechnological tools. An intensely blue-coloured pocilloporin, termed Rtms5, was expressed in Escherichia coli, purified and crystallized. Rtms5 was shown to be tetrameric, with deep blue crystals that diffract to 2.2 A resolution and belong to space group I4(1)22. The colour of this pocilloporin was observed to be sensitive to pH and a yellow (pH 3.5) and a red form (pH 4.5) of Rtms5 were also crystallized. These crystals belong to space group P4(2)22 and diffract to 2.4 A resolution or better.

The 2.2 A Crystal Structure of a Pocilloporin Pigment Reveals a Nonplanar Chromophore Conformation

Structure (London, England : 1993). Mar, 2003  |  Pubmed ID: 12623015

Reef-building corals contain host pigments, termed pocilloporins, that function to regulate the light environment of their resident microalgae by acting as a photoprotectant in excessive sunlight. We have determined the crystal structure of an intensely blue, nonfluorescent pocilloporin to 2.2 A resolution and a genetically engineered fluorescent variant to 2.4 A resolution. The pocilloporin chromophore structure adopts a markedly different conformation in comparison with the DsRed chromophore, despite the chromophore sequences (Gln-Tyr-Gly) being identical; the tyrosine ring of the pocilloporin chromophore is noncoplanar and in the trans configuration. Furthermore, the fluorescent variant adopted a noncoplanar chromophore conformation. The data presented here demonstrates that the conformation of the chromophore is highly dependent on its immediate environment.

The 2.0-A Crystal Structure of EqFP611, a Far Red Fluorescent Protein from the Sea Anemone Entacmaea Quadricolor

The Journal of Biological Chemistry. Nov, 2003  |  Pubmed ID: 12909624

We have crystallized and subsequently determined to 2.0-A resolution the crystal structure of eqFP611, a far red fluorescent protein from the sea anemone Entacmaea quadricolor. The structure of the protomer, which adopts a beta-can topology, is similar to that of the related monomeric green fluorescent protein (GFP). The quaternary structure of eqFP611, a tetramer exhibiting 222 symmetry, is similar to that observed for the more closely related red fluorescent protein DsRed and the chromoprotein Rtms5. The unique chromophore sequence (Met63-Tyr64-Gly65) of eqFP611, adopts a coplanar and trans conformation within the interior of the beta-can fold. Accordingly, the eqFP611 chromophore adopts a significantly different conformation in comparison to the chromophore conformation observed in GFP, DsRed, and Rtms5. The coplanar chromophore conformation and its immediate environment provide a structural basis for the far red, highly fluorescent nature of eqFP611. The eqFP611 structure extends our knowledge on the range of conformations a chromophore can adopt within closely related members of the green fluorescent protein family.

Training Nonhuman Primates Using Positive Reinforcement Techniques

Journal of Applied Animal Welfare Science : JAAWS. 2003  |  Pubmed ID: 14612264

FRET Reveals Changes in the F1-stator Stalk Interaction During Activity of F1F0-ATP Synthase

Biochimica Et Biophysica Acta. Dec, 2003  |  Pubmed ID: 14670607

A stator is proposed as necessary to prevent futile rotation of the F(1) catalytic sector of mitochondrial ATP synthase (mtATPase) during periods of ATP synthesis or ATP hydrolysis. Although the second stalk of mtATPase is generally believed to fulfil the role of a stator capable of withstanding the stress produced by rotation of the central rotor, there is little evidence to directly support this view. We show that interaction between two candidate proteins of the second stalk, OSCP and subunit b, fused at their C-termini to GFP variants and assembled into functional mtATPase can be monitored in mitochondria using fluorescence resonance energy transfer (FRET). Substitution of native OSCP with a variant containing a glycine 166 to asparagine (G166N) substitution yielded a metastable complex. In contrast to the enzyme containing native OSCP, FRET could be irreversibly lowered for the enzyme containing G166N at a rate that correlated closely with the rate of enzyme activity (ATP hydrolysis). The non-hydrolysable ATP analogue, AMP-PCP did not have this effect. We conclude that two candidate proteins of the stator stalk, OSCP and b, are subject to stresses during enzyme catalytic activity commensurate with their role as a part of a stator stalk.

Subunit Gamma-green Fluorescent Protein Fusions Are Functionally Incorporated into Mitochondrial F1F0-ATP Synthase, Arguing Against a Rigid Cap Structure at the Top of F1

The Journal of Biological Chemistry. Jan, 2003  |  Pubmed ID: 12414811

We have investigated the question of the presence of a cap structure located at the top of the F(1) alpha(3)beta(3) hexamer of the yeast mitochondrial F(1)F(0)-ATP synthase complex. Specifically, we sought to determine whether the putative cap has a rigid structure and occludes the central shaft space formed by the alpha(3)beta(3) hexamer or alternatively whether the cap is more flexible permitting access to the central shaft space under certain conditions. Thus, we sought to establish whether subunit gamma, an essential component of the F(1) central stalk housed within the central shaft space and whose N and C termini would both lie beneath a putative cap, could be fused at its C terminus to green fluorescent protein (GFP) without loss of enzyme function. The GFP moiety serves to report on the integrity and location of fusion proteins containing different length polypeptide linkers between GFP and subunit gamma, as well as being a potential occluding structure in itself. Functional incorporation of subunit gamma-GFP fusions into ATP synthase of yeast cells lacking native subunit gamma was demonstrated by the ability of intact complexes to hydrolyze ATP and retain sensitivity to oligomycin. Our conclusion is that the putative cap structure cannot be an inflexible structure, but must be of a more flexible nature consistent with the accommodation of subunit gamma-GFP fusions within functional ATP synthase complexes.

Cross-linking ATP Synthase Complexes in Vivo Eliminates Mitochondrial Cristae

Journal of Cell Science. May, 2004  |  Pubmed ID: 15126633

We have used the tetrameric nature of the fluorescent protein DsRed to cross-link F(1)F(O)-ATPase complexes incorporating a subunit gamma-DsRed fusion protein in vivo. Cells expressing such a fusion protein have impaired growth relative to control cells. Strikingly, fluorescence microscopy of these cells revealed aberrant mitochondrial morphology. Electron microscopy of cell sections revealed the absence of cristae and multiple layers of unfolded inner mitochondrial membrane. Complexes recovered from detergent lysates of mitochondria were present largely as tetramers. Co-expression of 'free' DsRed targeted to the mitochondria reduced F(1)F(O)-ATPase oligomerisation and partially reversed the impaired growth and abnormal mitochondrial morphology. We conclude that the correct arrangement of F(1)F(O)-ATPase complexes within the mitochondrial inner membrane is crucial for the genesis and/or maintenance of mitochondrial cristae and morphology. Our findings further suggest that F(1)F(O)-ATPase can exist in oligomeric associations within the membrane during respiratory growth.

Variations on the GFP Chromophore: A Polypeptide Fragmentation Within the Chromophore Revealed in the 2.1-A Crystal Structure of a Nonfluorescent Chromoprotein from Anemonia Sulcata

The Journal of Biological Chemistry. Jan, 2005  |  Pubmed ID: 15542608

We have determined to 2.1 A resolution the crystal structure of a dark state, kindling fluorescent protein isolated from the sea anemone, Anemonia sulcata. The chromophore sequence Met(63)-Tyr(64)-Gly(65) of the A. sulcata chromoprotein was previously proposed to comprise a 6-membered pyrazine-type heterocycle (Martynov, V. I., Savitsky, A. P., Martynova, N. Y., Savitsky, P. A., Lukyanov, K. A., and Lukyanov, S. A. (2001) J. Biol. Chem. 276, 21012-21016). However, our crystallographic data revealed the chromophore to comprise a 5-membered p-hydroxybenzylideneimidazolinone moiety that adopts a non-coplanar trans conformation within the interior of the GFP beta-can fold. Unexpectedly, fragmentation of the polypeptide was found to occur within the chromophore moiety, at the bond between Cys(62C) and Met(63N1.) Our structural data reveal that fragmentation of the chromophore represents an intrinsic, autocatalytic step toward the formation of the mature chromophore within the specific GFP-like proteins.

Guidance on the Transport of Laboratory Animals

Laboratory Animals. Jan, 2005  |  Pubmed ID: 15703122

The 2.1A Crystal Structure of the Far-red Fluorescent Protein HcRed: Inherent Conformational Flexibility of the Chromophore

Journal of Molecular Biology. May, 2005  |  Pubmed ID: 15876379

We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1A resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu(64)-Tyr(65)-Gly(66), was observed to adopt both a cis coplanar and a trans non-coplanar conformation. As a result of these two conformations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the beta-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible.

F1F0-ATP Synthase Complex Interactions in Vivo Can Occur in the Absence of the Dimer Specific Subunit E

Journal of Bioenergetics and Biomembranes. Apr, 2005  |  Pubmed ID: 15906150

Evidence suggests membrane bound F(1)F(0)-ATPase complexes form stable associations such that dimers can be retrieved from detergent lysates of mitochondria isolated from a range of sources including algae, higher plants, yeast and bovine heart, and plant chloroplasts. The physiological relevance of these interactions is not clear but may be connected with the formation and structure of mitochondrial cristae. We sought to demonstrate, in vivo, the association of F(1)F(0)-ATPases in yeast cells co-expressing two b subunits each fused at its C-terminus to a GFP variant appropriate for fluorescence resonance energy transfer (FRET; BFP as the donor and GFP as the acceptor fluorophore). Both subunit b-GFP and b-BFP fusions were assembled into functional complexes. FRET was observed from enzyme complexes in molecular proximity in respiring cells providing the first demonstration of the association, in vivo, of F(1)F(0)-ATPase complexes. Moreover, FRET was observed within cells lacking the dimer specific subunit e, indicating structured associations can occur within the inner membrane in the absence of subunit e.

Amino Acid Substitutions Around the Chromophore of the Chromoprotein Rtms5 Influence Polypeptide Cleavage

Biochemical and Biophysical Research Communications. Feb, 2006  |  Pubmed ID: 16414348

Extension of the conjugated pi-system of many all-protein chromophores with an acylimine bond is the basis for their red-shifted optical properties. The presence of this post-translational modification is evident in crystal structures of these proteins. Harsh denaturation of proteins containing an acylimine bond results in partial polypeptide cleavage. For the red fluorescent protein DsRed, the extent of cleavage is quantitative. However, this is not the case for the blue non-fluorescent chromoprotein Rtms5, even though all chromophores in tetrameric Rtms5 contain an acylimine bond. We have identified two positions around the chromophore of Rtms5 where substitutions can promote or suppress the extent of cleavage on harsh denaturation. We propose a model in which cleavage of Rtms5 is facilitated by a trans to cis isomerisation of the chromophore.

The 2.0 Angstroms Crystal Structure of a Pocilloporin at PH 3.5: the Structural Basis for the Linkage Between Color Transition and Halide Binding

Photochemistry and Photobiology. Mar-Apr, 2006  |  Pubmed ID: 16613486

The pocilloporin Rtms5 and an engineered variant Rtms5H146S undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK(a) values of 4.1 and 3.2 were determined for the blue (absorption lambda(max), 590 nm) to yellow (absorption lambda(max), approximately 453 nm) transitions of Rtms5 and Rtms5H146. The pK(a) for the blue-yellow transition of Rtms5H146S increased by 1.4 U in the presence of 0.1 M KI, whereas the pK(a) for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 angstroms resolution the crystal structure of a yellow form of Rtms5H146S at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5H146S structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5H146S and binding to halides.

The 2.1A Crystal Structure of CopGFP, a Representative Member of the Copepod Clade Within the Green Fluorescent Protein Superfamily

Journal of Molecular Biology. Jun, 2006  |  Pubmed ID: 16697009

The green fluorescent protein (avGFP), its variants, and the closely related GFP-like proteins are characterized structurally by a cyclic tri-peptide chromophore located centrally within a conserved beta-can fold. Traditionally, these GFP family members have been isolated from the Cnidaria although recently, distantly related GFP-like proteins from the Bilateria, a sister group of the Cnidaria have been described, although no representative structure from this phylum has been reported to date. We have determined to 2.1A resolution the crystal structure of copGFP, a representative GFP-like protein from a copepod, a member of the Bilateria. The structure of copGFP revealed that, despite sharing only 19% sequence identity with GFP, the tri-peptide chromophore (Gly57-Tyr58-Gly59) of copGFP adopted a cis coplanar conformation within the conserved beta-can fold. However, the immediate environment surrounding the chromophore of copGFP was markedly atypical when compared to other members of the GFP-superfamily, with a large network of bulky residues observed to surround the chromophore. Arg87 and Glu222 (GFP numbering 96 and 222), the only two residues conserved between copGFP, GFP and GFP-like proteins are involved in autocatalytic genesis of the chromophore. Accordingly, the copGFP structure provides an alternative platform for the development of a new suite of fluorescent protein tools. Moreover, the structure suggests that the autocatalytic genesis of the chromophore is remarkably tolerant to a high degree of sequence and structural variation within the beta-can fold of the GFP superfamily.

The 1.7 A Crystal Structure of Dronpa: a Photoswitchable Green Fluorescent Protein

Journal of Molecular Biology. Nov, 2006  |  Pubmed ID: 17010376

The green fluorescent protein (GFP), its variants, and the closely related GFP-like proteins possess a wide variety of spectral properties that are of widespread interest as biological tools. One desirable spectral property, termed photoswitching, involves the light-induced alteration of the optical properties of certain GFP members. Although the structural basis of both reversible and irreversible photoswitching events have begun to be unraveled, the mechanisms resulting in reversible photoswitching are less clear. A novel GFP-like protein, Dronpa, was identified to have remarkable light-induced photoswitching properties, maintaining an almost perfect reversible photochromic behavior with a high fluorescence to dark state ratio. We have crystallized and subsequently determined to 1.7 A resolution the crystal structure of the fluorescent state of Dronpa. The chromophore was observed to be in its anionic form, adopting a cis co-planar conformation. Comparative structural analysis of non-photoactivatable and photoactivatable GFPs, together with site-directed mutagenesis of a position (Cys62) within the Dronpa chromophore, has provided a basis for understanding Dronpa photoactivation. Specifically, we propose a model of reversible photoactivation whereby irradiation with light leads to subtle conformational changes within and around the environment of the chromophore that promotes proton transfer along an intricate polar network.

Endoplasmic Reticulum and Golgi Complex: Contributions To, and Turnover By, Autophagy

Traffic (Copenhagen, Denmark). Dec, 2006  |  Pubmed ID: 17040485

The degradation of cytoplasmic contents, especially organelles [mitochondria, peroxisomes, endoplasmic reticulum (ER), Golgi complex (GC)], cannot be accomplished solely by the cytosolic degradation machinery, of which the most prominent component is the proteasome. However, it is possible that such organelles (or portions thereof) can be degraded by the cell's autophagic machinery. In this manner, organelles can be either specifically or non-specifically targeted to the vacuole/lysosome for degradation. These processes can be triggered in response to different environmental cues. Here, we focus on two particular organelles, the ER and the GC, and their relationship with the autophagic process. Firstly, we briefly consider how these two organelles contribute to the synthesis and delivery of hydrolytic enzymes involved in autophagy as well as how they may potentially contribute to their own degradation by addressing the origin of the autophagic membrane. Secondly, we summarize the evidence for the turnover of these two organelles by autophagic processes in different organisms.

Recent Advances in All-protein Chromophore Technology

Biotechnology Annual Review. 2006  |  Pubmed ID: 17045191

The green fluorescent protein (GFP) is the foundation of a powerful technology that has revolutionized the way in which the life scientist carries out experiments in the living cell. The technology is continually evolving and improving through the development of existing proteins and discovery of new members of the all-protein chromophore (APC) family. This review gives an overview of the more recent advances in the technology with a particular focus on APCs having optical properties that are significantly red-shifted relative to those variants derived from Aequorea victoria GFP.

Electrospray Ionisation-cleavable Tandem Nucleic Acid Mass Tag-peptide Nucleic Acid Conjugates: Synthesis and Applications to Quantitative Genomic Analysis Using Electrospray Ionisation-MS/MS

Nucleic Acids Research. 2007  |  Pubmed ID: 17259215

The synthesis and characterization of isotopomer tandem nucleic acid mass tag-peptide nucleic acid (TNT-PNA) conjugates is described along with their use as electrospray ionisation-cleavable (ESI-Cleavable) hybridization probes for the detection and quantification of target DNA sequences by electrospray ionisation tandem mass spectrometry (ESI-MS/MS). ESI-cleavable peptide TNT isotopomers were introduced into PNA oligonucleotide sequences in a total synthesis approach. These conjugates were evaluated as hybridization probes for the detection and quantification of immobilized synthetic target DNAs using ESI-MS/MS. In these experiments, the PNA portion of the conjugate acts as a hybridization probe, whereas the peptide TNT is released in a collision-based process during the ionization of the probe conjugate in the electrospray ion source. The cleaved TNT acts as a uniquely resolvable marker to identify and quantify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed, quantitative DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

A Structural Basis for the PH-dependent Increase in Fluorescence Efficiency of Chromoproteins

Journal of Molecular Biology. May, 2007  |  Pubmed ID: 17376484

Within the fluorescent protein and chromoprotein family, the phenomenon of photoswitching is both intriguing and biotechnologically useful. Illumination of particular chromoproteins with intense light results in dramatic increases in fluorescence efficiency (termed kindling) and involves cis-trans isomerization of the chromophore. Here we report that chromophore isomerization can also be driven via alteration in pH. Specifically, we demonstrate that a number of naturally occurring chromoproteins, and their engineered variants, undergo a dramatic 20-100-fold increase in fluorescence efficiency at alkaline pH (>pH9.0). We have determined to 1.8 A resolution the structure of one such chromoprotein, Rtms5(H146S), in its highly far-red fluorescent form (Phi(F), 0.11 at pH 10.7) and compared it to the structure of the non-fluorescent form (Phi(F), 0.002 at pH 8.0). At high pH, the cyclic tri-peptide chromophore was observed to be mobile and distributed between a trans non-coplanar and a cis coplanar conformation, whereas at the lower pH, only a trans non-coplanar chromophore was observed. Calculation of pK(a) values suggested that titration of the side-chain of the conserved Glu215 close to the chromophore is involved in promoting the cis-coplanar conformation. Collectively, our data establish that isomerization to form a coplanar chromophore is a basis of the increased fluorescence efficiency at high pH. The phenomenon of pH-induced fluorescence gain has similarities with photoswitching, thereby providing a model to study the mechanism of kindling.

Autophagy and Vacuole Homeostasis: a Case for Self-degradation?

Autophagy. Sep-Oct, 2007  |  Pubmed ID: 17534141

The vacuole of yeast plays an important role in pH- and ion-homeostasis. Another important function of the vacuole, especially during nutrient deprivation, is the degradation of proteins, other macromolecules and organelles. To deliver these components into the vacuolar lumen, specific and sophisticated transport pathways such as autophagy have evolved. This review will first look at autophagy and its relationship to vacuole homeostasis, then move to the topic of vacuole self-degradation and possible reasons for its existence, and close by pointing very briefly to some areas for further research in these topics.

Different Fates of Mitochondria: Alternative Ways for Degradation?

Autophagy. Jan-Feb, 2007  |  Pubmed ID: 16929167

Cellular degradative processes including proteasomal and vacuolar/lysosomal (autophagic) degradation, as well as the activity of proteases (both cytosolic and mitochondrial), provide for a continuous turnover of damaged and obsolete macromolecules and organelles. Mitochondria are organelles essential for respiration and oxidative energy production in aerobic cells; they are also required for multiple biosynthetic pathways. As such, mitochondrial homeostasis is very important for cell survival. We review the evidence regarding the possible mechanisms for mitochondrial degradation. Increasingly, the evidence suggests autophagy plays a central role in the degradation of mitochondria. How mitochondria might be specifically selected for autophagy (mitophagy) remains an open question, although some evidence suggests that, under certain circumstances, in mammalian cells the Mitochondrial Permeability Transition (MPT) plays a role in initiation of the process. As more is learned about the functioning of autophagy as a degradation process, the greater the appreciation we are developing concerning its role in the control of mitochondrial degradation.

Rosella: a Fluorescent PH-biosensor for Reporting Vacuolar Turnover of Cytosol and Organelles in Yeast

Autophagy. Feb, 2008  |  Pubmed ID: 18094608

We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e., cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.

Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy in Higher Eukaryotes

Autophagy. Feb, 2008  |  Pubmed ID: 18188003

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Stimulation of Autophagy Suppresses the Intracellular Survival of Burkholderia Pseudomallei in Mammalian Cell Lines

Autophagy. Aug, 2008  |  Pubmed ID: 18483470

Burkholderia pseudomallei is the causative agent of melioidosis, a tropical infection of humans and other animals. The bacterium is an intracellular pathogen that can escape from endosomes into the host cytoplasm, where it replicates and infects adjacent cells. We investigated the role played by autophagy in the intracellular survival of B. pseudomallei in phagocytic and non-phagocytic cell lines. Autophagy was induced in response to B. pseudomallei invasion of murine macrophage (RAW 264.7) cells and a proportion of the bacteria co-localized with the autophagy effector protein LC3, a marker for autophagosome formation. Pharmacological stimulation of autophagy in RAW 264.7 and murine embryonic fibroblast (MEF) cell lines resulted in increased co-localization of B. pseudomallei with LC3 while basal levels of co-localization could be abrogated using inhibitors of the autophagic pathway. Furthermore, induction of autophagy decreased the intracellular survival of B. pseudomallei in these cell lines, but bacterial survival was not affected in MEF cell lines deficient in autophagy. Treatment of infected macrophages with chloramphenicol increased the proportion of bacteria within autophagosomes indicating that autophagic evasion is an active process relying on bacterial protein synthesis. Consistent with this hypothesis, we identified a B. pseudomallei type III secreted protein, BopA, which plays a role in mediating bacterial evasion of autophagy. We conclude that the autophagic pathway is a component of the innate defense system against invading B. pseudomallei, but which the bacteria can actively evade. However, when autophagy is pharmacologically induced using rapamycin, bacteria are actively sequestered in autophagosomes, ultimately decreasing their survival.

The Structure and Function of Mitochondrial F1F0-ATP Synthases

International Review of Cell and Molecular Biology. 2008  |  Pubmed ID: 18544496

We review recent advances in understanding of the structure of the F(1)F(0)-ATP synthase of the mitochondrial inner membrane (mtATPase). A significant achievement has been the determination of the structure of the principal peripheral or stator stalk components bringing us closer to achieving the Holy Grail of a complete 3D structure for the complex. A major focus of the field in recent years has been to understand the physiological significance of dimers or other oligomer forms of mtATPase recoverable from membranes and their relationship to the structure of the cristae of the inner mitochondrial membrane. In addition, the association of mtATPase with other membrane proteins has been described and suggests that further levels of functional organization need to be considered. Many reports in recent years have concerned the location and function of ATP synthase complexes or its component subunits on the external surface of the plasma membrane. We consider whether the evidence supports complete complexes being located on the cell surface, the biogenesis of such complexes, and aspects of function especially related to the structure of mtATPase.

Odorranalectin is a Small Peptide Lectin with Potential for Drug Delivery and Targeting

PloS One. 2008  |  Pubmed ID: 18584053

Lectins are sugar-binding proteins that specifically recognize sugar complexes. Based on the specificity of protein-sugar interactions, different lectins could be used as carrier molecules to target drugs specifically to different cells which express different glycan arrays. In spite of lectin's interesting biological potential for drug targeting and delivery, a potential disadvantage of natural lectins may be large size molecules that results in immunogenicity and toxicity. Smaller peptides which can mimic the function of lectins are promising candidates for drug targeting.

Monitoring Organelle Turnover in Yeast Using Fluorescent Protein Tags

Methods in Enzymology. 2008  |  Pubmed ID: 19185717

Refinements in Husbandry, Care and Common Procedures for Non-human Primates: Ninth Report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement

Laboratory Animals. Apr, 2009  |  Pubmed ID: 19286892

Preface Whenever animals are used in research, minimizing pain and distress and promoting good welfare should be as important an objective as achieving the experimental results. This is important for humanitarian reasons, for good science, for economic reasons and in order to satisfy the broad legal principles in international legislation. It is possible to refine both husbandry and procedures to minimize suffering and improve welfare in a number of ways, and this can be greatly facilitated by ensuring that up-to-date information is readily available. The need to provide such information led the British Veterinary Association Animal Welfare Foundation (BVAAWF), the Fund for the Replacement of Animals in Medical Experiments (FRAME), the Royal Society for the Prevention of Cruelty to Animals (RSPCA) and the Universities Federation for Animal Welfare (UFAW) to establish a Joint Working Group on Refinement (JWGR) in the UK. The chair is Professor David Morton and the secretariat is provided by the RSPCA. This report is the ninth in the JWGR series. The RSPCA is opposed to the use of animals in experiments that cause pain, suffering, distress or lasting harm and together with FRAME has particular concerns about the continued use of non-human primates. The replacement of primate experiments is a primary goal for the RSPCA and FRAME. However, both organizations share with others in the Working Group, the common aim of replacing primate experiments wherever possible, reducing suffering and improving welfare while primate use continues. The reports of the refinement workshops are intended to help achieve these aims. This report produced by the British Veterinary Association Animal Welfare Foundation (BVAAWF)/Fund for the Replacement of Animals in Medical Experiments (FRAME)/Royal Society for the Prevention of Cruelty to Animals (RSPCA)/Universities Federation for Animal Welfare (UFAW) Joint Working Group on Refinement (JWGR) sets out practical guidance on refining the husbandry and care of non-human primates (hereinafter primates) and on minimizing the adverse effects of some common procedures. It provides a valuable resource to help understand the physical, social and behavioural characteristics and needs of individual primates, and is intended to develop and complement the existing literature and legislative guidelines. Topics covered include refinements in housing, husbandry and common procedures such as restraint, identification and sampling, with comprehensive advice on issues such as primate communication, assessing and facilitating primate wellbeing, establishing and maintaining social groups, environmental and nutritional enrichment and animal passports. The most commonly used species are the key focus of this resource, but its information and recommendations are generally applicable to other species, provided that relevant individual species characteristics are taken into account.

Separation of Hexahistidine Fusion Proteins with Immobilized Metal Ion Affinity Chromatographic (IMAC) Sorbents Derived from M(N+)-tacn and Its Derivatives

Biotechnology and Bioengineering. Jul, 2009  |  Pubmed ID: 19350626

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7-triazacyclononane (tacn), bis(1,4,7-triazacyclononyl) ethane (dtne) and bis(1,4,7-triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu(2+), Ni(2+), Zn(2+), Mn(2+), Co(2+), and Cr(3+), for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C-terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S-transferase-Saccharomyces cerevisiae mitochondrial ATP synthase delta-subunit (GST-deltaATPase-His(6)), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)-nitrilotriacetic acid (im-Ni(2+)-NTA). Investigations using the recombinant GST-deltaATPase-His(6) and recombinant S. japonicum glutathione S-transferase (GST) lacking a hexahistidine tag have confirmed that the C-terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST-deltaATPase-His(6) can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu(2+) or Ni(2+), as IMAC systems.

SLIMMER (FHL1B/KyoT3) Interacts with the Proapoptotic Protein Siva-1 (CD27BP) and Delays Skeletal Myoblast Apoptosis

The Journal of Biological Chemistry. Sep, 2009  |  Pubmed ID: 19643733

The fhl1 gene encoding four-and-a-half LIM protein-1 (FHL1) and its spliced isoform, SLIMMER, is mutated in reducing body myopathy, X-linked myopathy with postural muscle atrophy, scapuloperoneal myopathy, and rigid spine syndrome. In this study we have identified a novel function for SLIMMER in delaying skeletal muscle apoptosis via an interaction with the proapoptotic protein Siva-1. Siva-1 was identified as a SLIMMER-specific-interacting protein using yeast two-hybrid screening, direct-binding studies, and glutathione S-transferase pulldown analysis of murine skeletal muscle lysates. In C2C12 skeletal myoblasts, SLIMMER and Siva co-localized in the nucleus; however, both proteins exhibited redistribution to the cytoplasm following the differentiation of mononucleated myoblasts to multinucleated myotubes. In sections of mature skeletal muscle from wild type mice, SLIMMER and Siva-1 co-localized at the Z-line. SLIMMER and Siva-1 were also enriched in Pax-7-positive satellite cells, muscle stem cells that facilitate repair and regeneration. Significantly, SLIMMER delayed Siva-1-dependent apoptosis in C2C12 myoblasts. In skeletal muscle sections from the mdx mouse model of Duchenne muscular dystrophy, SLIMMER and Siva-1 co-localized in the nucleus of apoptotic myofibers. Therefore, SLIMMER may protect skeletal muscle from apoptosis.

Proteomic Analysis of Embryonic Fasciola Hepatica: Characterization and Antigenic Potential of a Developmentally Regulated Heat Shock Protein

Veterinary Parasitology. Apr, 2010  |  Pubmed ID: 20089359

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.

Differential Proteomic Analysis of Arabidopsis Thaliana Genotypes Exhibiting Resistance or Susceptibility to the Insect Herbivore, Plutella Xylostella

PloS One. 2010  |  Pubmed ID: 20386709

A proteomic study was conducted to investigate physiological factors affecting feeding behaviour by larvae of the insect, Plutella xylostella, on herbivore-susceptible and herbivore-resistant Arabidopsis thaliana. The leaves of 162 recombinant inbred lines (Rils) were screened to detect genotypes upon which Plutella larvae fed least (P. xylostella-resistant) or most (P. xylostella-susceptible). 2D-PAGE revealed significant differences in the proteomes between the identified resistant and susceptible Rils. The proteomic results, together with detection of increased production of hydrogen peroxide in resistant Rils, suggest a correlation between P. xylostella resistance and the production of increased levels of reactive oxygen species (ROS), in particular H2O2, and that this was expressed prior to herbivory. Many of the proteins that were more abundant in the Plutella-resistant Rils are known in other biological systems to be involved in limiting ROS damage. Such proteins included carbonic anhydrases, malate dehydrogenases, glutathione S-transferases, isocitrate dehydrogenase-like protein (R1), and lipoamide dehydrogenase. In addition, patterns of germin-like protein 3 isoforms could also be indicative of higher levels of reactive oxygen species in the resistant Rils. Consistent with the occurrence of greater oxidative stress in the resistant Rils is the observation of greater abundance in susceptible Rils of polypeptides of the photosynthetic oxygen-evolving complex, which are known to be damaged under oxidative stress. The combined results suggest that enhanced production of ROS may be a major pre-existing mechanism of Plutella resistance in Arabidopsis, but definitive corroboration of this requires much further work.

Use of Granzyme B-based Fluorescent Protein Reporters to Monitor Granzyme Distribution and Granule Integrity in Live Cells

Biological Chemistry. Aug, 2010  |  Pubmed ID: 20536389

Reporter proteins comprising granzyme B (GrB) fused to eGFP, ecliptic pHluorin or mCherry, were generated and used to study granule (lysosome) distribution and properties in COS-1 cells and natural killer cells. The reporters resembled native GrB in biosynthesis and localization, and accumulated in granules. In live cells both the eGFP and pHluorin reporters were dark in lysosomes, but fluoresced when the granule integrity or pH was perturbed by Leu-Leu methyl ester, hydrogen peroxide, naphthazarin, or sphingosine treatment. By contrast, fluorescence of the mCherry reporter was not pH-dependent. The quenching of eGFP within granules indicates that this commonly-used fluorescent protein is not appropriate as a vital intra-lysosomal marker.

Welfare of the Minipig with Special Reference to Use in Regulatory Toxicology Studies

Journal of Pharmacological and Toxicological Methods. Nov-Dec, 2010  |  Pubmed ID: 20621655

This paper reviews the animal welfare challenges associated with the use of minipigs in toxicology testing, and compares these to published knowledge on the other widely used non-rodent species (dogs and non-human primates). Welfare challenges arise from housing and management of populations under laboratory conditions, and from the procedures carried out for product evaluation. Welfare assessment requires a multidisciplinary approach: cardiovascular parameters, adrenocortical hormones and behaviour are well known parameters. However, reliable non-invasive methods to assess welfare and species-specific benchmarks need further development in minipigs. Husbandry of minipigs (housing, diet, and socialisation needs) to promote good welfare is described in the revised Appendix A of the European Convention (ETS 123). This has been supplemented by knowledge of species biology and expert opinion from experienced minipig users. Challenges when using minipigs in toxicity testing have been reviewed in detail. Although deeper location of the peripheral blood vessels makes blood sampling more challenging, samples can be taken with minimal distress when staff members are well trained. Temporary and chronic vascular catheters can also be used for frequent sampling, and are likely to improve the welfare of the animals. Available training courses with a focus on stress free handling and dosing, as well as surgical placement of temporary and chronic vascular catheters, should be utilised to improve welfare during these procedures. Humane endpoints have been described, mainly based on current industry practices, but further scientific investigations are required. From an animal welfare perspective there are no basic restrictions to using minipigs in toxicity testing that are unique to this species. We conclude that it is easier to keep minipigs to a good standard of welfare under laboratory conditions than it is for dogs or non-human primates, since minipigs are not athletic (like dogs) or arboreal (like non-human primates).

Efficient Identification of Proteins from Ovaries and Hepatopancreas of the Unsequenced Edible Crab, Cancer Pagurus, by Mass Spectrometry and Homology-based, Cross-species Searching

Journal of Proteomics. Nov, 2010  |  Pubmed ID: 20656081

Proteome maps of hepatopancreas (midgut gland) and ovarian tissues of the crustacean, Cancer pagurus (Decapoda; edible crab) have been produced by 2D-PAGE and identification of proteins, following trypsin proteolysis, by electrospray MS/MS and database searching. Owing to the lack of sequence information on proteins and fully sequenced genomes amongst the decapod crustaceans and given the evolutionary distance to the nearest full genome database (Daphnia), it was necessary to adopt a non-conventional identification approach. Thus, a strategy was developed for effective identification of decapod proteins by sequence similarity, homology-based cross-species database searching, using various algorithms and a combination of NCBI Crustacea and Arthropoda databases, together with the Arthropoda PartiGene database (Blaxter, University of Edinburgh). In both hepatopancreas and ovary tissues, the largest group of proteins identified were a variety of enzymes, followed by a smaller number of storage/transport proteins [including vitellogenin (yolk protein), several subunits of hemocyanin, cryptocyanin, ferritin and calreticulin], with fewer structural proteins (actin, tubulin) and heat-shock proteins, in addition to a number of proteins of miscellaneous functions. Such protein identifications allow the development of tools, such as antibodies and RNA/DNA probes, to investigate the functions of the proteins in specific tissues during development.

Autophagy in Disease

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 20700706

Autophagy is a cellular quality control process by which cytoplasmic constituents including proteins, protein aggregates, organelles, and invading pathogens can be delivered to lysosomes for degradation. Autophagy is activated in response to changes in the internal status of the cell and/or changes in the extracellular environment. It is therefore essential for the maintenance of cellular homeostasis and for an efficient response to cellular stresses. As such autophagy has been implicated either in the pathogenesis, or response to a wide variety of diseases, bacterial, and viral infections, and ageing.

Mitophagy and Mitoptosis in Disease Processes

Methods in Molecular Biology (Clifton, N.J.). 2010  |  Pubmed ID: 20700707

Mitochondria play a very important role in cellular function, not only through key metabolic reactions and energy generation, but also by being a major site for production of reactive oxygen species and a key player in cell death. Therefore, mitochondrial dysfunction or damage may have severe consequences. Mitophagy (autophagic degradation of mitochondria) and mitoptosis (programmed destruction of mitochondria) are the processes by which cells can deal with impaired mitochondria. The efficiency of these processes may be a contributing factor to the pathogenesis of various diseases.

Comparative Proteomic Analysis of Triclabendazole Response in the Liver Fluke Fasciola Hepatica

Journal of Proteome Research. Oct, 2010  |  Pubmed ID: 20726552

Control of Fasciola hepatica infections of livestock in the absence of vaccines depends largely on the chemical triclabendazole (TCBZ) because it is effective against immature and adult parasites. Overdependence on a single drug and improper application is considered a significant factor in increasing global reports of fluke resistant to TCBZ. The mode(s) of action and biological target(s) of TCBZ are not confirmed, delaying detection and the monitoring of early TCBZ resistance. In this study, to further understand liver fluke response to TCBZ, the soluble proteomes of TCBZ-resistant and TCBZ-susceptible isolates of F. hepatica were compared with and without in vitro exposure to the metabolically active form of the parent drug triclabendazole sulphoxide (TCBZ-SO), via two-dimensional gel electrophoresis (2-DE). Gel image analysis revealed proteins displaying altered synthesis patterns and responses both between isolates and under TCBZ-SO exposure. These proteins were identified by mass spectrometry supported by a F. hepatica expressed sequence tag (EST) data set. The TCBZ responding proteins were grouped into three categories; structural proteins, energy metabolism proteins, and "stress" response proteins. This single proteomic investigation supported the reductionist experiments from many laboratories that collectively suggest TCBZ has a range of effects on liver fluke metabolism. Proteomics highlighted differences in the innate proteome profile of different fluke isolates that may influence future therapy and diagnostics design. Two of the TCBZ responding proteins, a glutathione transferase and a fatty acid binding protein, were cloned, produced as recombinants, and both found to bind TCBZ-SO at physiologically relevant concentrations, which may indicate a role in TCBZ metabolism and resistance.

Refinement of the Use of Food and Fluid Control As Motivational Tools for Macaques Used in Behavioural Neuroscience Research: Report of a Working Group of the NC3Rs

Journal of Neuroscience Methods. Nov, 2010  |  Pubmed ID: 20868708

This report provides practical guidance on refinement of the use of food and fluid control as motivational tools for macaques used in behavioural neuroscience research. The guidance is based on consideration of the scientific literature and, where data are lacking, expert opinion and professional experience, including that of the members of a Working Group convened by the United Kingdom National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). The report should be useful to researchers, veterinarians and animal care staff responsible for the welfare of macaques used in food and fluid control protocols, as well as those involved with designing, performing and analysing studies that use these protocols. It should also assist regulatory authorities and members of local ethical review processes or institutional animal care and use committees concerned with evaluating such protocols. The report provides a framework for refinement that can be tailored to meet local requirements. It also identifies data gaps and areas for future research and sets out the Working Group's recommendations on contemporary best practice.

The Intricacy of Nuclear Membrane Dynamics During Nucleophagy

Nucleus (Austin, Tex.). May-Jun, 2010  |  Pubmed ID: 21327066

The cell nucleus is an organelle bounded by a double-membrane which undergoes drastic reorganization during major cellular events such as cell division and apoptosis. Maintenance of proper nuclear structure, function and dynamics is central to organelle vitality. Over recent years growing evidence has shown that parts of the nucleus can be specifically degraded by an autophagic process termed nucleophagy. The process is best described in the yeast, Saccharomyces cerevisiae, where piecemeal microautophagy of the nucleus or nucleophagy (micronucleophagy) requires direct interaction of the nuclear membrane with that of the vacuole (the yeast lytic compartment). Here, we review the process of nucleophagy in the context of nuclear membrane dynamics, and examine the evidence for autophagic degradation of the nucleus in mammalian cells. Finally, we discuss the importance of nucleophagy as a 'housecleaning' mechanism for the nucleus under both normal and disease conditions.

The Burkholderia Pseudomallei Type III Secretion System and BopA Are Required for Evasion of LC3-associated Phagocytosis

PloS One. 2011  |  Pubmed ID: 21412437

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal infectious disease endemic in tropical regions worldwide, and especially prevalent in southeast Asia and northern Australia. This intracellular pathogen can escape from phagosomes into the host cytoplasm, where it replicates and infects adjacent cells. We previously demonstrated that, in response to B. pseudomallei infection of macrophage cell line RAW 264.7, a subset of bacteria co-localized with the autophagy marker protein, microtubule-associated protein light chain 3 (LC3), implicating autophagy in host cell defence against infection. Recent reports have suggested that LC3 can be recruited to both phagosomes and autophagosomes, thereby raising questions regarding the identity of the LC3-positive compartments in which invading bacteria reside and the mechanism of the autophagic response to B. pseudomallei infection. Electron microscopy analysis of infected cells demonstrated that the invading bacteria were either free in the cytosol, or sequestered in single-membrane phagosomes rather than double-membrane autophagosomes, suggesting that LC3 is recruited to B. pseudomallei-containing phagosomes. Partial or complete loss of function of type III secretion system cluster 3 (TTSS3) in mutants lacking the BopA (effector) or BipD (translocator) proteins respectively, resulted in delayed or no escape from phagosomes. Consistent with these observations, bopA and bipD mutants both showed a higher level of co-localization with LC3 and the lysosomal marker LAMP1, and impaired survival in RAW264.7 cells, suggesting enhanced killing in phagolysosomes. We conclude that LC3 recruitment to phagosomes stimulates killing of B. pseudomallei trapped in phagosomes. Furthermore, BopA plays an important role in efficient escape of B. pseudomallei from phagosomes.

V-ATPase Engagement in Autophagic Processes

Autophagy. Jun, 2011  |  Pubmed ID: 21494095

The proton pumping activity of V-ATPase is responsible for acidification of the lysosome/vacuole. The low lumenal pH of this organelle stimulates the activity of a battery of resident hydrolases responsible for the degradation of various nonselective and selective cargos delivered by autophagic processes. However, the role of V-ATPase in membrane dynamics required for the uptake of autophagic cargo is far from fully understood. Consideration of the available data leads us to speculate that autophagic processes involving direct membrane-to-membrane contacts between the selected cargo and the vacuolar membrane require functional V-ATPase.

The Molecular Action of the Novel Insecticide, Pyridalyl

Insect Biochemistry and Molecular Biology. Jul, 2011  |  Pubmed ID: 21497652

Pyridalyl is a recently discovered insecticide that exhibits high insecticidal activity against Lepidoptera and Thysanoptera. Pyridalyl action requires cytochrome P450 activity, possibly for production of a bioactive derivative, Pyridalyl metabolism being prevented by general P450 inhibitors. Apoptosis is apparently not involved in the cytotoxicity. Continuous culture of Spodoptera frugiperda Sf21 cells in sub-lethal doses of Pyridalyl, results in a Pyridalyl-resistant cell line. Probing the molecular action of Pyridalyl by comparison of the proteomes of Pyridalyl-resistant and -susceptible cell lines, revealed differential expression of a number of proteins, including the up-regulation of thiol peroxiredoxin (TPx), in the resistant cells. Treatment of Bombyx mori larvae with Pyridalyl, followed by comparison of the midgut microsomal sub-proteome, revealed the up-regulation of three proteasome subunits. Such subunits, together with Hsp70 stress proteins, glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) and thiol peroxiredoxin (TPx) were also up-regulated in the whole proteome of B. mori BM36 cells following treatment with the insecticide. The foregoing results lead to the hypothesis that cytochrome P450 action leads to an active Pyridalyl metabolite, which results in production of reactive oxygen species (ROS), that leads to damage to cellular macromolecules (e.g., proteins) and enhanced proteasome activity leads to increased protein degradation and necrotic cell death.

Microautophagy in Mammalian Cells: Revisiting a 40-year-old Conundrum

Autophagy. Jul, 2011  |  Pubmed ID: 21646866

The term microautophagy was first used in 1966 by de Duve and Wattiaux and subsequently applied, over the following two decades, to processes described in mammalian cells and involving the presence of lysosome-like organelles having multiple vesicles trapped in their lumen ("multivesicular lysosomes"). Concurrently, many studies suggested a view of microautophagy where the lysosomal membrane was either invaginated or projected arm-like protrusions to sequester cytosolic constituents into intralysosomal vesicles. Although microautophagy in mammalian cells has been traditionally considered as a form of autophagy constitutively active in the turnover of long-lived proteins, little is known about the mechanism and regulation of cargo selection. The lack of specific approaches to directly detect microautophagy in mammalian systems, aside from electron microscopy, is the major current limitation to addressing its physiological role(s) and possible contribution to particular disease states. In this review we consider the current state of knowledge about microautophagic processes. We examine some of the main characteristics of microautophagy in yeast with a view to assessing their relevance for our understanding of microautophagy in mammalian cells.

Role for the Burkholderia Pseudomallei Type Three Secretion System Cluster 1 BpscN Gene in Virulence

Infection and Immunity. Sep, 2011  |  Pubmed ID: 21768285

Burkholderia pseudomallei, the causal agent of melioidosis, employs a number of virulence factors during its infection of mammalian cells. One such factor is the type three secretion system (TTSS), which is proposed to mediate the transport and secretion of bacterial effector molecules directly into host cells. The B. pseudomallei genome contains three TTSS gene clusters (designated TTSS1, TTSS2, and TTSS3). Previous research has indicated that neither TTSS1 nor TTSS2 is involved in B. pseudomallei virulence in a hamster infection model. We have characterized a B. pseudomallei mutant lacking expression of the predicted TTSS1 ATPase encoded by bpscN. This mutant was significantly attenuated for virulence in a respiratory melioidosis mouse model of infection. In addition, analyses in vitro showed diminished survival and replication in RAW264.7 cells and an increased level of colocalization with the autophagy marker protein LC3 but an unhindered ability to escape from phagosomes. Taken together, these data provide evidence that the TTSS1 bpscN gene product plays an important role in the intracellular survival of B. pseudomallei and the pathogenesis of murine infection.

Vma8p-GFP Fusions Can Be Functionally Incorporated into V-ATPase, Suggesting Structural Flexibility at the Top of V1

International Journal of Molecular Sciences. 2011  |  Pubmed ID: 21845105

The vacuolar ATPase (V-ATPase) complex of yeast (Saccharomyces cerevisiae) is comprised of two sectors, V(1) (catalytic) and V(O) (proton transfer). The hexameric (A(3)B(3)) cylinder of V(1) has a central cavity that must accommodate at least part of the rotary stalk of V-ATPase, a key component of which is subunit D (Vma8p). Recent electron microscopy (EM) data for the prokaryote V-ATPase complex (Thermus thermophilus) suggest that subunit D penetrates deeply into the central cavity. The functional counterpart of subunit D in mitochondrial F(1)F(O)-ATP synthase, subunit γ, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit γ, we probed the location of the C-terminus of Vma8p by attachment of a large protein adduct, green fluorescent protein (GFP). We found that truncated Vma8p proteins lacking up to 40 C-terminal residues fused to GFP can be incorporated into functional V-ATPase complexes, and are able to support cell growth under alkaline conditions. We conclude that large protein adducts can be accommodated at the top of the central cavity of V(1) without compromising V-ATPase function, arguing for structural flexibility of the V(1) sector.

Strategies for Intracellular Survival of Burkholderia Pseudomallei

Frontiers in Microbiology. 2011  |  Pubmed ID: 22007185

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality that is prevalent in tropical regions of the world. A key component of the pathogenesis of melioidosis is the ability of B. pseudomallei to enter, survive, and replicate within mammalian host cells. For non-phagocytic cells, bacterial adhesins have been identified both on the bacterial surface and associated with Type 4 pili. Cell invasion involves components of one or more of the three Type 3 Secretion System clusters, which also mediate, at least in part, the escape of bacteria from the endosome into the cytoplasm, where bacteria move by actin-based motility. The mechanism of actin-based motility is not clearly understood, but appears to differ from characterized mechanisms in other bacterial species. A small proportion of intracellular bacteria is targeted by host cell autophagy, involving direct recruitment of LC3 to endosomes rather than through uptake by canonical autophagosomes. However, the majority of bacterial cells are able to circumvent autophagy and other intracellular defense mechanisms such as the induction of inducible nitric oxide synthase, and then replicate in the cytoplasm and spread to adjacent cells via membrane fusion, resulting in the formation of multi-nucleated giant cells. A potential role for host cell ubiquitin in the autophagic response to bacterial infection has recently been proposed.

Autophagy in Human Embryonic Stem Cells

PloS One. 2011  |  Pubmed ID: 22110659

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

Immunodetection of Granzyme B Tissue Distribution and Cellular Localisation

Methods in Molecular Biology (Clifton, N.J.). 2012  |  Pubmed ID: 22262447

Investigation of Granzyme B (GrB) function and pathophysiology in both human settings and rodent models increasingly involve the use of indirect immunofluorescence imaging and fluorescence-activated cell sorting, which requires reliable GrB antibodies that do not recognise other closely related granzymes. Here, we describe the validation (using a set of recombinant granzymes, and GrB-deficient cells) and application of widely available monoclonal antibodies to specifically monitor GrB in human or mouse cells.

Characterisation of the N'1 Isoform of the Cyclic AMP-dependent Protein Kinase (PK-A) Catalytic Subunit in the Nematode, Caenorhabditis Elegans

Archives of Biochemistry and Biophysics. Jan, 2012  |  Pubmed ID: 22286028

Multiple isoforms of the cyclic AMP-dependent protein kinase (PK-A) catalytic (C) subunit, arise as a consequence of the use of alternative splicing strategies during transcription of the kin-1 gene in the nematode, Caenorhabditis elegans. N-myristoylation is a common co-translational modification of mammalian PK-A C-subunits; however, the major isoform (N'3), originally characterised in C. elegans, is not N-myristoylated. Here, we show that N'1 isoforms are targets for N-myristoylation in C. elegans. We have demonstrated the in vivo incorporation of radioactivity into N'1 C-subunit isoforms, following incubation of nematodes with [(3)H]-myristic acid. HPLC and MALDI-TOF MS analysis of proteolytic digests of immunoprecipitates confirmed the presence of myristoyl-glycine in the C-subunit. In order to better understand the impact of the N'1 N-terminal sequence, and its myristoylation, on C-subunit activity, a chimerical C-subunit, consisting of the N'1 N-terminus from C. elegans and a murine core and C-terminal sequence was expressed. Myristoylation had no appreciable effect on the catalytic properties of the chimeric protein. However, the myristoylated chimeric protein did exhibit enhanced apolar targeting compared to the myristoylated wild-type murine polypeptide. This behaviour may reflect the inability of the N'1-encoded N-terminus sequence to correctly dock with a hydrophobic domain on the surface of the C-subunit.