Most proteins must fold into defined three-dimensional structures to gain functional activity. But in the cellular environment, newly synthesized proteins are at great risk of aberrant folding and aggregation, potentially forming toxic species. To avoid these dangers, cells invest in a complex network of molecular chaperones, which use ingenious mechanisms to prevent aggregation and promote efficient folding. Because protein molecules are highly dynamic, constant chaperone surveillance is required to ensure protein homeostasis (proteostasis). Recent advances suggest that an age-related decline in proteostasis capacity allows the manifestation of various protein-aggregation diseases, including Alzheimer's disease and Parkinson's disease. Interventions in these and numerous other pathological states may spring from a detailed understanding of the pathways underlying proteome maintenance.

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Molecular chaperones in protein folding and proteostasis

基礎講座 電気泳動(Electrophoresis)

MUCKE aims to mine a large volume of images, to structure them conceptually and to use this conceptual structuring in order to improve large-scale image retrieval. The last decade witnessed important progress concerning low-level image representations. However, there are a number problems which need to be solved in order to unleash the full potential of image mining in applications. The central problem with low-level representations is the mismatch between them and the human interpretation of image content. This problem can be instantiated, for instance, by the incapability of existing descriptors to capture spatial relationships between the concepts represented or by their incapability to convey an explanation of why two images are similar in a content-based image retrieval framework. We start by assessing existing local descriptors for image classification and by proposing to use co-occurrence matrices to better capture spatial relationships in images. The main focus in MUCKE is on cleaning large scale Web image corpora and on proposing image representations which are closer to the human interpretation of images. Consequently, we introduce methods which tackle these two problems and compare results to state of the art methods. Note: some aspects of this deliverable are withheld at this time as they are pending review. Please contact the authors for a preview.

http://ieeexplore.ieee.org/iel2/4524/12820/00592460.pdf

Image Processing

The interferon (IFN) system is an extremely powerful antiviral response that is capable of controlling most, if not all, virus infections in the absence of adaptive immunity. However, viruses can still replicate and cause disease in vivo, because they have some strategy for at least partially circumventing the IFN response. We reviewed this topic in 2000 [Goodbourn, S., Didcock, L. & Randall, R. E. (2000). J Gen Virol 81, 2341-2364] but, since then, a great deal has been discovered about the molecular mechanisms of the IFN response and how different viruses circumvent it. This information is of fundamental interest, but may also have practical application in the design and manufacture of attenuated virus vaccines and the development of novel antiviral drugs. In the first part of this review, we describe how viruses activate the IFN system, how IFNs induce transcription of their target genes and the mechanism of action of IFN-induced proteins with antiviral action. In the second part, we describe how viruses circumvent the IFN response. Here, we reflect upon possible consequences for both the virus and host of the different strategies that viruses have evolved and discuss whether certain viruses have exploited the IFN response to modulate their life cycle (e.g. to establish and maintain persistent/latent infections), whether perturbation of the IFN response by persistent infections can lead to chronic disease, and the importance of the IFN system as a species barrier to virus infections. Lastly, we briefly describe applied aspects that arise from an increase in our knowledge in this area, including vaccine design and manufacture, the development of novel antiviral drugs and the use of IFN-sensitive oncolytic viruses in the treatment of cancer.

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures.

The biological functions of proteins are governed by their three-dimensional fold. Protein folding, maintenance of proteome integrity, and protein homeostasis (proteostasis) critically depend on a complex network of molecular chaperones. Disruption of proteostasis is implicated in aging and the pathogenesis of numerous degenerative diseases. In the cytosol, different classes of molecular chaperones cooperate in evolutionarily conserved folding pathways. Nascent polypeptides interact cotranslationally with a first set of chaperones, including trigger factor and the Hsp70 system, which prevent premature (mis)folding. Folding occurs upon controlled release of newly synthesized proteins from these factors or after transfer to downstream chaperones such as the chaperonins. Chaperonins are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. This review focuses on recent advances in understanding the mechanisms of chaperone action in promoting and regulating protein folding and on the pathological consequences of protein misfolding and aggregation.

Molecular Chaperone Functions in Protein Folding and Proteostasis

The influenza virus RNA segment 8 encodes two proteins, NS1 and NS2, by differential splicing. The collinear transcript acts as mRNA for NS1 protein, while the spliced mRNA encodes NS2 protein. The splicing of NS1 mRNA was studied in cells transfected with a recombinant plasmid that has the cDNA of RNA segment 8 cloned under the SV40 late promoter and polyadenylation signals. As described for influenza virus-infected cells, NS1 mRNA was poorly spliced to yield NS2 mRNA. However, inactivation of the NS1 gene, but not the NS2 gene, led to a substantial increase in the splicing efficiency, as shown by the relative accumulations of NS1 and NS2 mRNAs. This effect was not specific for NS1 mRNA, since the splicing of the endogenous SV40 early transcript was altered in such a way that t-Ag mRNA was almost eliminated. These changes in the splicing pattern coincided with a strong inhibition of the mRNA nucleocytoplasmic transport. Both NS1 and NS2 mRNAs were retained in the nucleus of cells expressing NS1 protein, but no effect was observed when only NS2 protein was expressed. Furthermore, other mRNAs tested, such as T-Ag mRNA and the non-spliceable nucleoprotein transcript, were also retained in the nucleus upon expression of NS1 protein, suggesting that it induced a generalized block of mRNA export from the nucleus.

https://www.embopress.org/doi/pdf/10.1002/j.1460-2075.1994.tb06310.x

Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.

The effect of NS1 protein on the efficiency of influenza virus mRNA translation was evaluated by determining the accumulation of nucleoprotein (NP) or M1 mRNAs in the cytoplasm of cells expressing either of these genes alone or in combination with the NS1 gene, as well as the total cell accumulation of NP or M1 protein. Coexpression of NS1, but not of NS2 protein, led to increases in the translation of these mRNAs in the range of 5- to 100-fold. This translation enhancement was specific for viral mRNAs, since the translation of neither cat nor lacZ mRNAs was affected by the coexpression of NS1 protein. The use of chimeric cat genes containing the 5'-extracistronic sequences of the influenza virus mRNAs corresponding to segment 2, 7, or 8 indicated that these sequences can in part account for the observed effect. The enhancement of viral mRNA translation mediated by NS1 protein was due to an increase in the translation initiation rate, since the sizes of NP-specific polysomes, but not those of lacZ-specific polysomes, was significantly higher in cells coexpressing NS1 protein than in those expressing only the NP gene.

Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs.

The establishment and maintenance of asymmetries in certain cells implies the localized expression of many proteins, a property often enhanced by the localization of the corresponding mRNAs (for reviews, see references 2 and 47). The relevance of mRNA localization at precise sites of the cell in the definition of polarity of developing embryos has been documented in both Drosophila melanogaster and Xenopus laevis. Thus, the positions of gurken and oskar mRNAs in the fly oocyte define its dorsoventral axis (36) and the location of the pole plasm at the posterior pole (15), respectively. Likewise, the localization of the bicoid and nanos mRNAs at the anterior and posterior poles of the embryo, respectively, leads to the generation of two opposing gradients of their protein products and ultimately to the definition of the head, thorax, and abdomen of the embryo (50). In the case of X. laevis, several mRNAs, such as Xcat2, Xcat3, and Xlsirt, are directed to the germ plasm (34), while others, such as Vg1 and Xwnt11, accumulate at the vegetal cortex (30). By analogy to the D. melanogaster genes, these mRNAs are thought to play a role in defining patterns in the X. laevis embryo. In fact, a region of the Xcat2 protein shows sequence homology to the zinc finger domain of nanos protein (34).

The specific localization of certain mRNAs to precise sites within the cell is not an exclusive property of germ cells or developing embryos. A number of observations indicate that some of the mRNAs of somatic cells are also localized at different sites within the cell. Thus, myelin-binding protein is translated in oligodendrocytes from free ribosomes on localized mRNA (51), and the microtubule-associated protein MAP2 is translated preferentially in dendrites of the neurons (23). Likewise, different actin protein isoforms are translated from their mRNAs at distinct sites of myoblasts (27).

In some cases, the intracellular localization of mRNAs involves cis-acting signals consisting of distinct secondary structures of their untranslated regions (UTRs) (reviewed in reference 47). These cis signals are thought to mediate localization by interaction with RNA-binding proteins, the best studied of which is the Staufen protein of D. melanogaster (dmStaufen) (48; reviewed in reference 50). dmStaufen protein is the product of a maternal mRNA of D. melanogaster that is involved in the accumulation of bicoid and oskar mRNAs at the anterior pole of the embryo and the posterior pole of the oocyte, respectively (48). It contains double-stranded RNA (dsRNA)-binding domains that associate with bicoid mRNA through a precise secondary structure in its 3′ UTR (18).

Our group has been studying the influenza virus nonstructural protein NS1, an RNA-binding protein (26, 33, 42) that may be involved in several regulatory processes during viral infection (reviewed in reference 37). These include the modulation of pre-mRNA splicing (20, 21, 32), the retention of poly(A)-containing RNA in the nucleus (20, 42), and the stimulation of viral mRNA translation (10, 14). These properties of NS1 protein seem related to particular interactions with certain cellular or viral RNA molecules and may also involve interaction with specific cellular factors (26, 40, 42, 43). In the course of a genetic screen to detect candidate cellular proteins that may pertain to these biochemical effects, we identified a human gene with homology to the dmStaufen gene. The encoded protein (hStaufen-like) was localized by immunofluorescence to the rough endoplasmic reticulum in cultured cells, was associated with polysomes, and behaved like an RNA-binding protein.

A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.

https://jb.asm.org/content/jb/186/5/1337.full.pdf

The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds.

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 A 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.

Ana Beloso

Papers

Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.

Influenza virus NS1 protein enhances the rate of translation initiation of viral mRNAs.

A Human Sequence Homologue of Staufen Is an RNA-Binding Protein That Is Associated with Polysomes and Localizes to the Rough Endoplasmic Reticulum

The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds.

Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin.