A Foerster Resonance Energy Transfer (FRET)-based System Provides Insight into the Ordered Assembly of Yeast Septin Hetero-octamers [Protein Structure and Folding]

September 28th, 2015 by Booth, E. A., Vane, E. W., Dovala, D., Thorner, J.

Prior studies in both budding yeast (Saccharomyces cerevisiae) and in human cells have established that septin protomers assemble into linear hetero-octameric rods with two-fold rotational symmetry. In mitotically-growing yeast cells, five septin subunits are expressed (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) and assemble into two types of rods that differ only in their terminal subunit: Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1. EM analysis has shown that, under low-salt conditions, the Cdc11-capped rods polymerize end-to-end to form long paired filaments, whereas Shs1-capped rods form arcs, spirals and rings. To develop a facile method to study septin polymerization in vitro, we exploited our previous work where we generated septin complexes in which all endogenous cysteine (Cys) residues were eliminated by site-directed mutagenesis, except an introduced E294C mutation in Cdc11 in these experiments. Mixing samples of a preparation of such single-Cys containing Cdc11-capped rods that have been separately derivatized with organic dyes that serve as donor and acceptor, respectively, for FRET provided a spectroscopic method to monitor filament assembly mediated by Cdc11-Cdc11 interaction and to measure its affinity under specified conditions. Modifications of this same FRET scheme also allow us to assess whether Shs1-capped rods are capable of end-to-end association either with themselves or with Cdc11-capped rods. This FRET approach also was used to follow the binding to septin filaments of a septin-interacting protein, the type II myosin-binding protein Bni5.
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Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain [Molecular Bases of Disease]

September 28th, 2015 by

Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5'-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP) bound crystal structure that informs a multi-step model for binding single-stranded DNA. Overall, these high-resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis.

Characterization of Two Human Skeletal Calsequestrin Mutants Implicated in Malignant Hyperthermia and Vacuolar Aggregates Myopathy [Protein Structure and Folding]

September 28th, 2015 by Lewis, K. M., Ronish, L. A., Rios, E., Kang, C.

Calsequestrin 1 (hCasq1) is the principal Ca2+ storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates, while its M87T variant is weakly associated with malignant hyperthermia (MH). We characterized the consequences of these mutations with studies of the human proteins in vitro. Equilibrium dialysis and turbidity measurements showed that D244G and, to a lesser extent, M87T partially lose Ca2+ binding exhibited by wild type hCasq1 at high Ca2+ concentrations. D244G aggregates abruptly and abnormally, a property that fully explains the protein inclusions that characterize its phenotype. D244G crystallized in low Ca2+ concentrations lacks two Ca2+ ions normally present in wild type, which weakens the hydrophobic core of Domain II. D244G crystallized in high Ca2+ concentrations regains its missing ions and Domain II order, but shows a novel dimeric interaction. The M87T mutation causes a major shift of the α-helix bearing the mutated residue, significantly weakening the back-to-back interface essential for tetramerization. D244G exhibited the more severe structural and biophysical property changes, which matches the different pathophysiological impacts of these mutations.
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The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells Without a Signal Peptide at Hand? [Cell Biology]

September 28th, 2015 by

For long, protein transport into the extracellular space was believed to strictly depend on signal peptide mediated translocation into the lumen of the endoplasmic reticulum. More recently, this view has been challenged and the molecular mechanisms of unconventional secretory processes are beginning to emerge. Here, we focus on unconventional secretion of fibroblast growth factor 2 (FGF2), a secretory mechanism that is based upon direct protein translocation across plasma membranes. Through a combination of genome-wide RNAi screening approaches and biochemical reconstitution experiments, the basic machinery of FGF2 secretion was identified and validated. This includes the integral membrane protein ATP1A1, the phosphoinositide PI(4,5)P2, Tec kinase as well as membrane proximal heparan sulfate proteoglycans on cell surfaces. Hallmarks of unconventional secretion of FGF2 are (i) Sequential molecular interactions with the inner leaflet along with Tec kinase dependent tyrosine phosphorylation of FGF2, (ii) PI(4,5)P2-dependent oligomerization and membrane pore formation and (iii) Extracellular trapping of FGF2 mediated by heparan sulfate proteoglycans on cell surfaces. Here we discuss new developments regarding this process including the mechanism of FGF2 oligomerization during membrane pore formation, the functional role of ATP1A1 in FGF2 secretion and the possibility that other proteins secreted by unconventional means make use of a similar mechanism to reach the extracellular space. Furthermore, given the prominent role of extracellular FGF2 in tumor induced angiogenesis, we will discuss possibilities to develop highly specific inhibitors of FGF2 secretion, a novel approach that may give way for lead compounds with a high potential to develop into anti-cancer drugs.

The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and is Disrupted in Cancer [Cell Biology]

September 28th, 2015 by

The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A K119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1 protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1 and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1 which generate a composite ubiquitin binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving, (i) the ubiquitin carboxyl hydrolase (UCH) catalytic domain of BAP1 which interacts with the hydrophobic patch of ubiquitin and (ii) the CTD domain which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI, and notably an in frame deletion in the CTD that inhibits its interaction with ASXL1/2, DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of BAP1/ASXL2 axis might contribute to cancer development.

The Relationship Between Glycan-Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-Forming Toxin [Membrane Biology]

September 28th, 2015 by

Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell-surface receptors that increase the local toxin concentration on cell membranes thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. While it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell-lines we show that VCC is unlikely to utilize high-affinity protein receptors like structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino-acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan-binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding-interactions.
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Rotenone-induced Impairment of Mitochondrial Electron Transport Chain Confers a Selective Priming Signal for NLRP3 Inflammasome Activation [Immunology]

September 28th, 2015 by Won, J.-H., Park, S., Hong, S., Son, S., Yu, J.-W.

Mitochondrial dysfunction is considered crucial for NLRP3 inflammasome activation partly through its release of mitochondrial toxic products such as mitochondrial ROS (mROS) and mitochondrial DNA (mtDNA). While previous studies have shown that classical NLRP3-activating stimulations lead to mROS generation and mtDNA release, it remains poorly understood whether and how mitochondrial damage-derived factors may contribute to NLRP3 inflammasome activation. Here, we demonstrate that impairment of the mitochondrial electron transport chain by rotenone licenses NLRP3 inflammasome activation only upon costimulation with ATP, but not with nigericin or alum. Rotenone-induced priming of NLRP3 in the presence of ATP triggered the formation of speck-like NLRP3 or ASC aggregates and the association of NLRP3 with ASC, resulting in NLRP3-dependent caspase-1 activation. Mechanistically, rotenone confers a priming signal for NLRP3 inflammasome activation only in the context of aberrant high-grade, but not low-grade, mROS production and mitochondrial hyperpolarization. By contrast, rotenone/ATP-mediated mtDNA release and mitochondrial depolarization are likely to be merely an indication of mitochondrial damage rather than triggering factors for NLRP3 inflammasome activation. Our results provide a molecular insight into the selective contribution made by mitochondrial dysfunction to the NLRP3 inflammasome pathway.
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Truncated Amyloid-{beta}(11-40/42) from Alzheimer’s Disease Binds Copper2+ with a Femtomolar Affinity and Influences Fibre Assembly [Molecular Biophysics]

September 25th, 2015 by Barritt, J. D., Viles, J. H.

Alzheimer's disease (AD) coincides with the formation of extracellular amyloid plaques composed of the amyloid-β (Aβ) peptide. Aβ is typically forty residues long (Aβ(1-40)) but can have variable C- and N- termini. Naturally occurring N-terminally truncated Aβ(11-40/42) is found in the cerebrospinal fluid and has a similar abundance to Aβ(1-42), constituting one fifth of the plaque load. Based on its specific N-terminal sequence we hypothesized that truncated Aβ(11-40/42) would have an elevated affinity for Cu2+. Various spectroscopic techniques, complimented with transmission electron microscopy, were used to determine the properties of the Cu2+Aβ(11-40/42) interaction and how Cu2+ influences amyloid fibre formation. We show, Cu2+Aβ(11-40) forms a tetragonal complex with a 34 ± 5 femtomolar dissociation constant at pH 7.4. This affinity is three orders of magnitude tighter than Cu2+ binding to Aβ(1-40/42) and more than an order of magnitude tighter than that of serum albumin, the extracellular Cu2+ transport protein. Furthermore, Aβ(11-40/42) forms fibres twice as fast as Aβ(1-40) with a very different morphology; forming bundles of very short amyloid rods. Substoichiometric Cu2+ drastically perturbs Aβ(11-40/42) assembly, stabilizing much longer fibres. The very tight femtomolar affinity of Cu2+ for Aβ(11-40/42) explains the high levels of Cu2+ observed in AD plaques.
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IL-10 mediated immunosuppression: March-I induction regulates antigen presentation by macrophages but not dendritic cells [Immunology]

September 25th, 2015 by Mittal, S. K., Cho, K.-J., Ishido, S., Roche, P. A.

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of DCs and macrophages to stimulate antigen-specific CD4 T cells, however the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin-ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of APC function in macrophages is March-I-dependent whereas in DCs suppression is March-I-independent.

Plant Protochlorophyllide Oxidoreductases A and B – Catalytic Efficiency and Initial Reaction Steps [Plant Biology]

September 25th, 2015 by

The enzyme protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) has a key role in plant development. It catalyzes one of the later steps in chlorophyll synthesis, the light-induced reduction of protochlorophyllide (PChlide) into chlorophyllide (Chlide) in the presence of NADPH. Two isozymes of plant POR, POR A and POR B from barley, which differ in their function during plant life, are compared with respect to their substrate binding affinity, catalytic efficiency, and catalytic mechanism. POR B as compared to POR A shows an 5-fold higher binding affinity for protochlorophyllide a (PChlide) and an about 6-fold higher catalytic efficiency measured as ratio kcat/KM. Based on the reaction intermediates, which can be trapped at low temperatures the same reaction mechanism operates in both POR A and POR B. In contrast to results reported for POR enzymes from cyanobacteria, the initial light-driven step, which occurs at temperatures below 180 K already involves the full chemistry of the photoreduction and yields the reaction product, Chlide, in an enzyme-bound form. The subsequent dark reactions, which include cofactor (NADP+) release and cofactor (NADPH) rebinding, show different temperature dependencies for POR A and POR B and suggest a higher conformational flexibility of POR B in the surrounding of the active center. Both the higher substrate binding affinity and well-adapted enzyme dynamics are held responsible for the increased catalytic activity of POR B as compared to POR A.