The lipid side of unfolded protein response.

Although our current knowledge of the molecular crosstalk between the ER stress, the unfolded protein response (UPR), and lipid homeostasis remains limited, there is increasing evidence that dysregulation of either protein or lipid homeostasis profoundly affects the other. Most research regarding UPR signaling in human diseases has focused on the causes and consequences of disrupted protein folding. The UPR itself consists of very complex pathways that function to not only maintain protein homeostasis, but just as importantly, modulate lipid biogenesis to allow the ER to adjust and promote cell survival. Lipid dysregulation is known to activate many aspects of the UPR, but the complexity of this crosstalk remains a major research barrier. ER lipid disequilibrium and lipotoxicity are known to be important contributors to numerous human pathologies, including insulin resistance, liver disease, cardiovascular diseases, neurodegenerative diseases, and cancer. Despite their medical significance and continuous research, however, the molecular mechanisms that modulate lipid synthesis during ER stress conditions, and their impact on cell fate decisions, remain poorly understood. Here we summarize the current view on crosstalk and connections between altered lipid metabolism, ER stress, and the UPR.

Replication stress response in fission yeast differentially depends on maintaining proper levels of Srs2 helicase and Rrp1, Rrp2 DNA translocases.

Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome.

Ubiquitin-Dependent and Independent Proteasomal Degradation in Host-Pathogen Interactions.

Ubiquitin, a small protein, is well known for tagging target proteins through a cascade of enzymatic reactions that lead to protein degradation. The ubiquitin tag, apart from its signaling role, is paramount in destabilizing the modified protein. Here, we explore the complex role of ubiquitin-mediated protein destabilization in the intricate proteolysis process by the 26S proteasome. In addition, the significance of the so-called ubiquitin-independent pathway and the role of the 20S proteasome are considered. Next, we discuss the ubiquitin-proteasome system's interplay with pathogenic microorganisms and how the microorganisms manipulate this system to establish infection by a range of elaborate pathways to evade or counteract host responses. Finally, we focus on the mechanisms that rely either on (i) hijacking the host and on delivering pathogenic E3 ligases and deubiquitinases that promote the degradation of host proteins, or (ii) counteracting host responses through the stabilization of pathogenic effector proteins.

E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein.

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

Beyond K48 and K63: non-canonical protein ubiquitination.

Protein ubiquitination has become one of the most extensively studied post-translational modifications. Originally discovered as a critical element in highly regulated proteolysis, ubiquitination is now regarded as essential for many other cellular processes. This results from the unique features of ubiquitin (Ub) and its ability to form various homo- and heterotypic linkage types involving one of the seven different lysine residues or the free amino group located at its N-terminus. While K48- and K63-linked chains are broadly covered in the literature, the other types of chains assembled through K6, K11, K27, K29, and K33 residues deserve equal attention in the light of the latest discoveries. Here, we provide a concise summary of recent advances in the field of these poorly understood Ub linkages and their possible roles in vivo.

The ancillary N-terminal region of the yeast AP-1 transcription factor Yap8 contributes to its DNA binding specificity.

Activator protein 1 (AP-1) is one of the largest families of basic leucine zipper (bZIP) transcription factors in eukaryotic cells. How AP-1 proteins achieve target DNA binding specificity remains elusive. In Saccharomyces cerevisiae, the AP-1-like protein (Yap) family comprises eight members (Yap1 to Yap8) that display distinct genomic target sites despite high sequence homology of their DNA binding bZIP domains. In contrast to the other members of the Yap family, which preferentially bind to short (7-8 bp) DNA motifs, Yap8 binds to an unusually long DNA motif (13 bp). It has been unclear what determines this unique specificity of Yap8. In this work, we use molecular and biochemical analyses combined with computer-based structural design and molecular dynamics simulations of Yap8-DNA interactions to better understand the structural basis of DNA binding specificity determinants. We identify specific residues in the N-terminal tail preceding the basic region, which define stable association of Yap8 with its target promoter. We propose that the N-terminal tail directly interacts with DNA and stabilizes Yap8 binding to the 13 bp motif. Thus, beside the core basic region, the adjacent N-terminal region contributes to alternative DNA binding selectivity within the AP-1 family.

Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein.

Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

Identification of the Elusive Pyruvate Reductase of Chlamydomonas reinhardtii Chloroplasts.

Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD(+)-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.

Insights into the relationship between the haem-binding pocket and the redox potential of c6 cytochromes: four atomic resolution structures of c6 and c6-like proteins from Synechococcus sp. PCC 7002.

The structure of cytochrome c6C from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.03 Šresolution. This is the first structural report on the recently discovered cyanobacterial cytochrome c6-like proteins found in marine and nitrogen-fixing cyanobacteria. Despite high similarity in the overall three-dimensional fold between cytochromes c6 and c6C, the latter shows saliently different electrostatic properties in terms of surface charge distribution and dipole moments. Its midpoint redox potential is less than half of the value for typical c6 cytochromes and results mainly from the substitution of one residue in the haem pocket. Here, high-resolution crystal structures of mutants of both cytochromes c6 and c6C are presented, and the impact of the mutation of specific residues in the haem-binding pocket on the redox potential is discussed. These findings contribute to the elucidation of the structure-function relationship of c6-like cytochromes.

Anchoring a plant cytochrome P450 via PsaM to the thylakoids in Synechococcus sp. PCC 7002: evidence for light-driven biosynthesis.

Plants produce an immense variety of specialized metabolites, many of which are of high value as their bioactive properties make them useful as for instance pharmaceuticals. The compounds are often produced at low levels in the plant, and due to their complex structures, chemical synthesis may not be feasible. Here, we take advantage of the reducing equivalents generated in photosynthesis in developing an approach for producing plant bioactive natural compounds in a photosynthetic microorganism by functionally coupling a biosynthetic enzyme to photosystem I. This enables driving of the enzymatic reactions with electrons extracted from the photosynthetic electron transport chain. As a proof of concept, we have genetically fused the soluble catalytic domain of the cytochrome P450 CYP79A1, originating from the endoplasmic reticulum membranes of Sorghum bicolor, to a photosystem I subunit in the cyanobacterium Synechococcus sp. PCC 7002, thereby targeting it to the thylakoids. The engineered enzyme showed light-driven activity both in vivo and in vitro, demonstrating the possibility to achieve light-driven biosynthesis of high-value plant specialized metabolites in cyanobacteria.

Crystal structure of CyanoQ from the thermophilic cyanobacterium Thermosynechococcus elongatus and detection in isolated photosystem II complexes.

The PsbQ-like protein, termed CyanoQ, found in the cyanobacterium Synechocystis sp. PCC 6803 is thought to bind to the lumenal surface of photosystem II (PSII), helping to shield the Mn4CaO5 oxygen-evolving cluster. CyanoQ is, however, absent from the crystal structures of PSII isolated from thermophilic cyanobacteria raising the possibility that the association of CyanoQ with PSII might not be a conserved feature. Here, we show that CyanoQ (encoded by tll2057) is indeed expressed in the thermophilic cyanobacterium Thermosynechococcus elongatus and provide evidence in support of its assignment as a lipoprotein. Using an immunochemical approach, we show that CyanoQ co-purifies with PSII and is actually present in highly pure PSII samples used to generate PSII crystals. The absence of CyanoQ in the final crystal structure is possibly due to detachment of CyanoQ during crystallisation or its presence in sub-stoichiometric amounts. In contrast, the PsbP homologue, CyanoP, is severely depleted in isolated PSII complexes. We have also determined the crystal structure of CyanoQ from T. elongatus to a resolution of 1.6 Å. It lacks bound metal ions and contains a four-helix up-down bundle similar to the ones found in Synechocystis CyanoQ and spinach PsbQ. However, the N-terminal region and extensive lysine patch that are thought to be important for binding of PsbQ to PSII are not conserved in T. elongatus CyanoQ.

Crystal structure of the Psb28 accessory factor of Thermosynechococcus elongatus photosystem II at 2.3 Å.

Members of the Psb28 family of proteins are accessory factors implicated in the assembly and repair of the photosystem II complex. We present here the crystal structure of the Psb28 protein (Tlr0493) found in the thermophilic cyanobacterium Thermosynechococcus elongatus at a resolution of 2.3 Å. Overall the crystal structure of the Psb28 monomer is similar to the solution structures of C-terminally His-tagged Psb28-1 from Synechocystis sp. PCC 6803 obtained previously by nuclear magnetic resonance spectroscopy. One new aspect is that Escherichia coli-expressed T. elongatus Psb28 is able to form dimers in solution and packs as a dimer of dimers in the crystal. Analysis of wild type and mutant strains of Synechocystis 6803 by blue native-polyacrylamide gel electrophoresis suggests that Psb28-1, the closest homologue to T. elongatus Psb28 in this organism, also exists as an oligomer in vivo, most likely a dimer. In line with the prediction based on the crystal structure of T. elongatus Psb28, the addition of a 3× Flag-tag to the C-terminus of Synechocystis 6803 Psb28-1 interferes with the accumulation of the Psb28-1 oligomer in vivo. In contrast, the more distantly related Psb28-2 protein found in Synechocystis 6803 lacks the residues that stabilize dimer formation in the T. elongatus Psb28 crystal and is detected as a monomer in vivo. Overall our data suggest that the dimer interface in the Psb28 crystal might be physiologically relevant.

Structure of the RuBisCO chaperone RbcX from the thermophilic cyanobacterium Thermosynechococcus elongatus.

The crystal structure of TeRbcX, a RuBisCO assembly chaperone from the cyanobacterium Thermosynechococcus elongatus, a thermophilic organism, has been determined at 1.7 Šresolution. TeRbcX has an unusual cysteine residue at position 103 that is not found in RbcX proteins from mesophilic organisms. Unlike wild-type TeRbcX, a mutant protein with Cys103 replaced by Ala (TeRbcX-C103A) could be readily crystallized. The structure revealed that the overall fold of the TeRbcX homodimer is similar to those of previously crystallized RbcX proteins. Normal-mode analysis suggested that TeRbcX might adopt an open or closed conformation through a hinge movement pivoted on a kink in two long α4 helices. This type of conformational transition is presumably connected to RbcL (the large RuBisCO subunit) binding during the chaperone function of the RuBisCO assembly.

Atomic-resolution structure of reduced cyanobacterial cytochrome c6 with an unusual sequence insertion.

The structure of the reduced form of cytochrome c(6) from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.2 A and refined to an R-factor of 0.107. This protein is unique among all known cytochromes c(6), owing to the presence of an unusual seven-residue insertion, KDGSKSL(44-50), which differs from the insertion found in the recently discovered plant cytochromes c(6A). Furthermore, the present protein is unusual because of its very high content (36%) of the smallest residues (glycine and alanine). The structure reveals that the overall fold of the protein is similar to that of other class I c-type cytochromes, despite the presence of the specific insertion. The insertion is located within the most variable region of the cytochrome c(6) sequence, i.e. between helices II and III. The first six residues [KDGSKS(44-49)] form a loop, whereas the last residue, Leu50, extends the N-terminal beginning of helix III. Several specific noncovalent interactions are found inside the insertion, as well as between the insertion and the rest of the protein. The crystal structure contains three copies of the cytochrome c(6) molecule per asymmetric unit, and is characterized by an unusually high packing density, with solvent occupying barely 17.58% of the crystal volume.

Deeply branching c6-like cytochromes of cyanobacteria.

The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.