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SUMMARY: CCT is required to regulate interorganelle contact sites through kinesin‐1 dynamics, modulating peroxisome and mitochondria activity, and ultimately controlling lipid cell content and metabolism. Cell proteostasis includes gene transcription, protein translation, folding of de novo proteins, post‐translational modifications, secretion, degradation and recycling. By profiling the proteome of extracellular vesicles (EVs) from T cells, we have found the chaperonin complex CCT, involved in the correct folding of particular proteins. By limiting CCT cell‐content by siRNA, cells undergo altered lipid composition and metabolic rewiring towards a lipid‐dependent metabolism, with increased activity of peroxisomes and mitochondria. This is due to dysregulation of the dynamics of interorganelle contacts between lipid droplets, mitochondria, peroxisomes and the endolysosomal system. This process accelerates the biogenesis of multivesicular bodies leading to higher EV production through the dynamic regulation of microtubule‐based kinesin motors. These findings connect proteostasis with lipid metabolism through an unexpected role of CCT. [ABSTRACT FROM AUTHOR]

Activity‐regulated cytoskeleton‐associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self‐assembly into virus‐like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self‐association and capsid formation is largely unknown. Here, we identified a 28‐amino‐acid stretch in the mammalian Arc N‐terminal (NT) domain that is necessary and sufficient for self‐association. Within this region, we identified a 7‐residue oligomerization motif, critical for the formation of virus‐like capsids. Purified wild‐type Arc formed capsids as shown by transmission and cryo‐electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic‐resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled‐coil interface, strongly supporting NT‐NT domain interactions in Arc oligomerization. The NT coil–coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc‐facilitated endocytosis. Furthermore, using single‐molecule photobleaching, we show that Arc mRNA greatly enhances higher‐order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self‐association above the dimer stage, mRNA‐induced oligomerization, and formation of virus‐like capsids. Database: The coordinates and structure factors for crystallographic analysis of the oligomerization region were deposited at the Protein Data Bank with the entry code 6YTU. [ABSTRACT FROM AUTHOR]

Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril's morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4-methylaminophenyl benzothiazole and thioflavin- S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54-63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54- 63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts. [ABSTRACT FROM AUTHOR]

G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1-Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1-Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate. [ABSTRACT FROM AUTHOR]

The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/ lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran. [ABSTRACT FROM AUTHOR]

ABSTRACT The eukaryotic cytosolic chaperonin CCT is a molecular machine involved in assisting the folding of proteins involved in important cellular processes. Like other chaperonins, CCT is formed by a double-ring structure but, unlike all of them, each ring is composed of eight different, albeit homologous subunits. This complexity has probably to do with the specificity in substrate interaction and with the mechanism of protein folding that takes place during the chaperonin functional cycle, but its detailed molecular basis remains unknown. We have analyzed the known proteomes in search of residues that are differentially conserved in the eight subunits, as predictors of functional specificity (specificity-determining positions; SDPs). We have found that most of these SDPs are located near the ATP binding site, and that they define four CCT clusters, corresponding to subunits CCT3, CCT6, CCT8 and CCT1/2/4/5/7. Our results point to a spatial organisation of the CCT subunits in two opposite areas of the ring and provide a molecular explanation for the previously described asymmetry in the hydrolysis of ATP. Proteins 2014; 82:703-707. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php. [ABSTRACT FROM AUTHOR]

The membrane proximal external region (MPER) of the fusogenic HIV-1 glycoprotein-41 harbors the epitope sequence recognized by 2F5, a broadly neutralizing antibody isolated from an infected individual. Structural mimicry of the conserved MPER 2F5 epitope constitutes a pursued goal in the field of anti-HIV vaccine development. It has been proposed that 2F5 epitope folding into its native state is attained in the vicinity of the membrane interface and might involve interactions with other viral structures. Here we present results indicating that oligomeric complexes established between MPER and the conserved amino-terminal fusion peptide (FP) can partition into lipid vesicles and be specifically bound by the 2F5 antibody at their surfaces. Cryo-transmission electron microscopy of liposomes doped with MPER:FP peptide mixtures provided the structural grounds for complex recognition by antibody at lipid bilayer surfaces. Supporting the immunogenicity of the membrane-bound complex, these MPER:FP peptide-vesicle formulations could trigger cross-reactive anti-MPER antibodies in rabbits. Thus, our observations suggest that contacts with N-terminal regions of gp41 may stabilize the 2F5 epitope as a membrane-surface antigen. [ABSTRACT FROM AUTHOR]

DNA electrostatic character is mostly determined by both water and counterions activities in the phosphate backbone, which together with base sequence, further confer its higher order structure. The authors overstretch individual double-stranded DNA molecules in water-ethanol solutions to investigate the modulation of its mechanical stability by hydration and polycations. The authors found that DNA denatures as ethanol concentration is increased and spermine concentration decreased. This is manifested by an increase in melting hysteresis between the stretch and release curves, with sharp transition at 10% ethanol and reentrant behavior at 60%, by a loss of cooperativity in the overstretching transition and by a dramatic decrease of both the persistence length and the flexural rigidity. Changes in base-stacking stability which are characteristic of the B-A transition between 70 and 80% ethanol concentration do not manifest in the mechanical properties of the double-helical molecule at low or high force or in the behavior of the overstretching and melting transitions within this ethanol concentration range. This is consistent with a mechanism in which A-type base-stacking is unstable in the presence of tension. Binding of motor proteins to DNA locally reduces the number of water molecules and therefore, our results may shed light on analogous reduced-water activity of DNA conditions caused by other molecules, which interact with DNA in vivo. © 2011 Wiley Periodicals, Inc. Biopolymers 97: 199-208, 2012. [ABSTRACT FROM AUTHOR]

Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and β-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin. [ABSTRACT FROM AUTHOR]

Symmetrical cell division requires duplication of DNA and protein content to generate two daughter cells. Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood. We describe an alternative splice form of SadB encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action through phosphorylation of γ-tubulin on Ser 131, as expression of a phosphomimetic Ser 131-to-Asp γ-tubulin mutant alone increased centrosome numbers, whereas non-phosphorylatable Ala 131-γ-tubulin impaired centrosome duplication. We propose that SADB kinase activity controls centrosome homeostasis by regulating phosphorylation of γ-tubulin. [ABSTRACT FROM AUTHOR]

The influenza viruses contain a segmented, single-stranded RNA genome of negative polarity. Each RNA segment is encapsidated by the nucleoprotein and the polymerase complex into ribonucleoprotein particles (RNPs), which are responsible for virus transcription and replication. Despite their importance, information about the structure of these RNPs is scarce. We have determined the three-dimensional structure of a biologically active recombinant RNP by cryo-electron microscopy. The structure shows a nonameric nucleoprotein ring (at 12 Å resolution) with two monomers connected to the polymerase complex (at 18 Å resolution). Docking the atomic structures of the nucleoprotein and polymerase domains, as well as mutational analyses, has allowed us to define the interactions between the functional elements of the RNP and to propose the location of the viral RNA. Our results provide the first model for a functional negative-stranded RNA virus ribonucleoprotein complex. The structure reported here will serve as a framework to generate a quasi-atomic model of the molecular machine responsible for viral RNA synthesis and to test new models for virus RNA replication and transcription. [ABSTRACT FROM AUTHOR]

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo. [ABSTRACT FROM AUTHOR]

Chaperones, a group of proteins that assist the folding of other proteins, seem to work in a coordinated manner. Two major chaperone families are heat-shock protein families Hsp60 and Hsp70. Here we show for the first time the formation of a stable complex between chaperonin-containing TCP-1 (CCT) and Hsc70, two eukaryotic representatives of these chaperone families. This interaction takes place between the apical domain of the CCTβ subunit and the nucleotide binding domain of Hsc70, and may serve to deliver the unfolded substrate from Hsc70 to the substrate binding region of CCT. We also show that a similar interaction does not occur between their prokaryotic counterparts GroEL and DnaK, suggesting that in eukarya the two types of chaperones have evolved to a concerted action that makes the folding task more efficient. [ABSTRACT FROM AUTHOR]

The eukaryotic cytosolic chaperonin CCT (chaperonin containing TCP-1) is the most complex of all chaperonins—an oligomeric structure built from two identical rings, each composed of single copies of eight different subunits. The arrangement of the eight subunits within each ring has been characterised for some time, but the phasing between the two rings remains unknown. Here, three-dimensional reconstructions generated by cryoelectron microscopy of complexes between CCT and either of two different monoclonal antibodies that react specifically with the CCTɛ and CCTδ subunits have been used to determine the phasing between the two chaperonin rings. The inter-ring arrangement is such that up/down inter-ring communication always involves two different CCT subunits in all eight positions, and the group of subunits concerned with the initiation and completion of the folding cycle cluster together both in the intra- and inter-ring arrangement. This supports a sequential mechanism of conformational changes between the two interacting rings. [ABSTRACT FROM AUTHOR]

A long-standing paradox in the study of T cell antigen recognition is that of the high specificity-low affinity T cell receptor (TCR)-major histocompatibility complex peptide (MHCp) interaction. The existence of multivalent TCRs could resolve this paradox because they can simultaneously improve the avidity observed for monovalent interactions and allow for cooperative effects. We have studied the stoichiometry of the TCR by Blue Native-polyacrylamide gel electrophoresis and found that the TCR exists as a mixture of monovalent (αβγε) and multivalent complexes with two or more ligand-binding TCRα/(&B subunits. The coexistence of monovalent and multivalent complexes was confirmed by electron microscopy after label fracture of intact T cells, thus ruling out any possible artifact caused by detergent solubilization. We found that although only the multivalent complexes become phosphorylated at low antigen doses, both multivalent and monovalent TCRs are phosphorylated at higher doses. Thus, the multivalent TCRs could be responsible for sensing low concentrations of antigen, whereas the monovalent TCRs could be responsible for dose-response effects at high concentrations, conditions in which the multivalent TCRs are saturated. Thus, besides resolving TCR stoichiometry, these data can explain how T cells respond to a wide range of MHCp concentrations while maintaining high sensitivity. [ABSTRACT FROM AUTHOR]

The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD. [ABSTRACT FROM AUTHOR]

Chaperonins are large oligomers made up of two superimposed rings, each enclosing a cavity used for the folding of other proteins. Among the chaperonins, the eukaryotic cytosolic chaperonin CCT is the most complex, not only with regard to its subunit composition but also with respect to its function, still not well understood. Unlike the more well studied eubacterial chaperonin GroEL, which binds any protein that presents stretches of hydrophobic residues, CCT recognises in its substrates specific binding determinants and interacts with them through particular combinations of CCT subunits. Folding then occurs after the conformational changes induced in the chaperonin upon nucleotide binding have occurred, through a mechanism that, although still poorly defined, clearly differs from the one established for GroEL. Although CCT seems to be mainly involved in the folding of actin and tubulin, other substrates involved in various cellular roles are beginning to be characterised, including many WD40-repeat, 7-blade propeller proteins. [Copyright &y& Elsevier]

Folding to completion of actin and tubulin in the eukaryotic cytosol requires their interaction with cytosolic chaperonin CCT [containing tailless complex polypeptide 1 (TCP-1). Three-dimensional reconstructions of nucleotide-free CCT complexed to either actin or tubulin show that CCT stabilizes both cytoskeletal proteins in open and quasi-folded con-formations mediated through interactions that are both subunit specific and geometry dependent. Here we find that upon ATP binding, mimicked by the non-hydrolysable analog AMP-PNP (5'-adenylylimido-diphosphate), to both CCT-α-actin and CCT- β-tubulin complexes, the chaperonin component undergoes concerted movements of the apical domains, resulting in the cavity being closed off by the helical protrusions of the eight apical domains. However, in contrast to the GroE system, generation of this closed state does not induce the release of the substrate into the chaperonin cavity, and both cytoskeletal proteins remain bound to the chaperonin apical domains. Docking of the AMP-PNP-CCT-bound con-formations of a-actin and β-tubulin to their respective native atomic structures suggests that both proteins have progressed towards their native states. [ABSTRACT FROM AUTHOR]

A three-dimensional structural model of an influenza virus ribonucleoprotein particle reconstituted in vivo from recombinant proteins and a model genomic vRNA has been generated by electron microscopy. It shows a circular shape and contains nine nucleoprotein monomers, two of which are connected with the polymerase complex. The nucleoprotein monomers show a curvature that may be responsible for the formation of helical structures in the full-size viral ribonucleoproteins. The monomers show distinct contact boundaries at the two sides of the particle, suggesting that the genomic RNA may be located in association with the nucleoprotein at the base of the ribonucleoprotein complex. Sections of the three-dimensional model show a trilobular morphology in the polymerase complex that is consistent with the presence of its three subunits. [ABSTRACT FROM AUTHOR]

Three-dimensional reconstruction from cryoelectron micrographs of the eukaryotic cytosolic chaperonin CCT complexed to tubulin shows that CCT interacts with tubulin (both the α and β isoforms) using five specific CCT subunits. The CCT-tubulin interaction has a different geometry to the CCT-actin interaction, and a mixture of shared and unique CCT subunits is used in binding the two substrates. Docking of the atomic structures of both actin and tubulin to their CCT-bound conformation suggests a common mode of chaperonin-substrate interaction. CCT stabilizes quasi-native structures in both proteins that are open through their domain-connecting hinge regions, suggesting a novel mechanism and function of CCT in assisted protein folding. [ABSTRACT FROM AUTHOR]

Herein, we describe the synthesis of highly emissive amphiphilic N-annulated PBI 1 decorated with oligo ethylene glycol (OEG) side chains. These polar side chains allow the straightforward solubility of 1 in solvents of different polarity such as water, iPrOH, dioxane, or chloroform. Compound 1 self-assembles in aqueous media by π-stacking of the aromatic units and van der Waals interactions, favored by the hydrophobic effect. The hypo- and hypsochromic effect observed in the UV-Vis spectra of 1 in water in comparison to chloroform is diagnostic of H-type aggregation. Solvent denaturation experiments allow deriving the free Gibbs energy for the self-assembly process in aqueous media and the factor m that is indicative of the influence exerted by a good solvent in the stability of the final aggregates. The ability of compound 1 to self-assemble in water yields globular aggregates that have been visualized by TEM imaging. [ABSTRACT FROM AUTHOR]

In this work, the successful preparation and characterization of gold nanorods (AuNRs) coated with a mesoporous silica shell (AuNRs@Simes) was achieved. Conjugation with methylene blue (MB) as a model drug using ultrasound-stimulated loading has been explored for further application in light-mediated antibacterial studies. Lyophilization of this conjugated nanosystem was analyzed using trehalose (TRH) as a cryogenic protector. The obtained stable dry formulation shows potent antimicrobial activity against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria after a simple post-treatment irradiation method with a red laser during a short time period. [ABSTRACT FROM AUTHOR]