The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies [Microbiology]

February 5th, 2019 by Eric Amado, Gunther Muth, Ignacio Arechaga, Elena Cabezon

TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces. Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation.

15-Deoxy-{Delta}12,14-prostaglandin J2 promotes phosphorylation of eukaryotic initiation factor 2{alpha} and activates the integrated stress response [Neurobiology]

February 5th, 2019 by Devin Tauber, Roy Parker

Stress granules (SGs) are cytoplasmic RNA-protein aggregates formed in response to inhibition of translation initiation. SGs contribute to the stress response and are implicated in a variety of diseases including cancer and some forms of neurodegeneration. Neurodegenerative diseases often involve chronic phosphorylation of eukaryotic initiation factor 2α (eIF2α), with deletions of eIF2α kinases or treatment with eiF2α kinase inhibitors being protective in some animal models of disease. However, how and why the integrated stress response (ISR) is activated in different forms of neurodegeneration remains unclear. Since neuroinflammation is common to many neurodegenerative diseases, we hypothesized that inflammatory factors contribute to ISR activation in a cell non-autonomous manner. Using fluorescence microscopy and immunoblotting, we show here that the endogenously produced product of inflammation, 15-Deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2), triggers eIF2α phosphorylation thereby activating the ISR, repressing bulk translation, and triggering stress granule formation. Our findings define a mechanism by which inflammation activates the ISR in a cell non-autonomous manner and suggest that inhibition of 15-d-PGJ2 production might be a useful therapeutic strategy in some neuroinflammatory contexts.
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A stable tetramer is not the only oligomeric state that mitochondrial single-stranded DNA binding proteins can adopt. [DNA and Chromosomes]

January 7th, 2019 by Saurabh P Singh, Vandna Kukshal, Roberto Galletto

Mitochondrial single-stranded DNA binding proteins (mtSSBs) are required for mitochondrial DNA replication and stability and are generally assumed to form homo-tetramers, and this species is proposed to be the one active for ssDNA binding. However, we recently reported that the mtSSB from Saccharomyces cerevisiae (ScRim1) forms homo-tetramers at high protein concentrations, whereas at low protein concentrations, it dissociates into dimers that bind ssDNA with high affinity . In this work, using a combination of analytical ultracentrifugation techniques and DNA binding experiments with fluorescently labeled DNA oligonucleotides, we tested whether the ability of ScRim1 to form dimers is unique among mtSSBs. Whereas human mtSSBs and those from Schizosaccharomyces pombe, Xenopus laevis and Xenopus tropicalis formed stable homo-tetramers, the mtSSBs from Candida albicans and Candida parapsilosis formed stable homo-dimers. Moreover, the mtSSBs from Candida nivariensis and Candida castellii formed tetramers at high protein concentrations, whereas at low protein concentrations they formed dimers, as did ScRim1. Mutational studies revealed that the ability to form either stable tetramers or dimers depended on a complex interplay of more than one amino acid at the dimer dimer interface and the C-terminal unstructured tail. In conclusion, our findings indicate that mtSSBs can adopt different oligomeric states, ranging from stable tetramers to stable dimers, and suggest that a dimer of mtSSB may be a physiologically relevant species that binds to ssDNA in some yeast species.

Post-transcriptional regulation of the Pseudomonas aeruginosa heme assimilation system (Has) fine-tunes extracellular heme sensing [Gene Regulation]

December 28th, 2018 by Alecia T. Dent, Susana Mourino, Weiliang Huang, Angela Wilks

Pseudomonas aeruginosa is an opportunistic pathogen that utilizes heme as a primary iron source within the host. Extracellular heme is sensed via a heme assimilation system (has) that encodes an extra cytoplasmic function (ECF) σ factor system. Herein, using has deletion mutants, qPCR analyses, and immunoblotting, we show that the activation of the σ factor HasI requires heme release from the hemophore HasAp to the outer membrane receptor HasR. Using RT-PCR and 5’-RACE, we observed that following transcriptional activation of the co-transcribed hasRAp, it is further processed into specific mRNAs varying in stability. We noted that the processing and variation in stability of the hasAp and hasR mRNAs in response to heme provides a mechanism for differential expression from co-transcribed genes. The multiple layers of post-transcriptional regulation of the ECF signaling cascade, including the previously reported post-transcriptional regulation of HasAp by the heme metabolites biliverdin IXβ and IXδ, allows fine tuning of the cell surface signaling system in response to extracellular heme levels. We hypothesize that the complex post-transcriptional regulation of the Has system provides P. aeruginosa an advantage in colonizing a variety of physiological niches in the host.
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Friend or foe — post-translational modifications as regulators of phase separation and RNP granule dynamics [Molecular Bases of Disease]

December 26th, 2018 by Mario Hofweber, Dorothee Dormann

Ribonucleoprotein (RNP) granules are membrane-less organelles consisting of RNA-binding proteins (RBPs) and RNA. RNA granules form through liquid–liquid phase separation (LLPS), whereby weak promiscuous interactions among RBPs and/or RNAs create a dense network of interacting macromolecules and drive the phase separation. Post-translational modifications (PTMs) of RBPs have emerged as important regulators of LLPS and RNP granule dynamics, as they can directly weaken or enhance the multivalent interactions between phase-separating macromolecules or can recruit or exclude certain macromolecules into or from condensates. Here, we review recent insights into how PTMs regulate phase separation and RNP granule dynamics, in particular arginine (R)-methylation and phosphorylation. We discuss how these PTMs regulate the phase behavior of prototypical RBPs and how, as “friend or foe”, they might influence the assembly, disassembly, or material properties of cellular RNP granules, such as stress granules or amyloid-like condensates. We particularly highlight how PTMs control the phase separation and aggregation behavior of disease-linked RBPs. We also review how disruptions of PTMs might be involved in aberrant phase transitions and the formation of amyloid-like protein aggregates as observed in neurodegenerative diseases.

Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex [Protein Synthesis and Degradation]

December 17th, 2018 by Janakiram R Vangala, Senthil K Radhakrishnan

Inhibition of the proteasome leads to proteotoxic stress which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNA interference screen in an engineered cell line reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for Nrf1’s transcriptional activity. Given that RUVBL1 is part of different multi-subunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60-Nrf1 axis to alter proteasome function in various human diseases including cancer.

Structure of human cortisol-producing cytochrome P450 11B1 bound to the breast cancer drug fadrozole provides insights for drug design [Molecular Bases of Disease]

November 13th, 2018 by Simone Brixius-Anderko, Emily E. Scott

Human cytochrome P450 11B1 (CYP11B1) is responsible for the final step generating the steroid hormone cortisol, which controls stress and immune responses and glucose homeostasis. CYP11B1 is a promising drug target to manage Cushing’s disease, a disorder arising from excessive cortisol production. However, the design of selective inhibitors has been hampered because structural information for CYP11B1 is unavailable and the enzyme has high amino acid sequence identity (93%) to a closely related enzyme, the aldosterone-producing CYP11B2. Here we report the X-ray crystal structure of human CYP11B1 (at 2.1 Å resolution) in complex with fadrozole, a racemic compound normally used to treat breast cancer by inhibiting estrogen-producing CYP19A1. Comparison of fadrozole-bound CYP11B1 with fadrozole-bound CYP11B2 revealed that despite conservation of the active site residues, overall structures and active sites had structural rearrangements consistent with distinct protein functions and inhibition. While fadrozole binds to both CYP11B enzymes by coordinating the heme iron, CYP11B2 binds to the R enantiomer of fadrozole, whereas CYP11B1 binds to the S enantiomer, each with distinct orientations and interactions. These results provide insights into the cross-reactivity of drugs across multiple steroidogenic cytochrome P450 enzymes, provide a structural basis for understanding human steroidogenesis, and pave the way for the design of more selective inhibitors of both human CYP11B enzymes.
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Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus [Cell Biology]

November 13th, 2018 by Kingkamon Junkunlo, Kenneth Soderhall, Irene Soderhall

Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and non-enzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish Pacifastacus leniusculus. We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT as well as from guinea pig in a concentration-dependent manner. Cystamine injection could decrease TGase activity in HPT without affecting production of reactive oxygen species (ROS). Moreover the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.

Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches [Enzymology]

November 13th, 2018 by Shinpei Uno, Hironori Kimura, Masatoshi Murai, Hideto Miyoshi

NADH-quinone oxidoreductase (respiratory complex I) couples NADH-to-quinone electron transfer to the translocation of protons across the membrane. Even though the architectures of the quinone-access channel in the enzyme have been modeled by X-ray crystallography and cryo-EM, conflicting findings raise the question whether the models fully reflect physiologically relevant states present throughout the catalytic cycle. To gain further insights into the structural features of the binding pocket for quinone/inhibitor, we performed chemical biology experiments using bovine heart sub-mitochondrial particles. We synthesized ubiquinones that are oversized (SF-UQs) or lipid-like (PC-UQs) and are highly unlikely to enter and transit the predicted narrow channel. We found that SF-UQs and PC-UQs can be catalytically reduced by complex I, albeit only at moderate or low rates. Moreover, quinone-site inhibitors completely blocked the catalytic reduction and the membrane potential formation coupled to this reduction. Photoaffinity-labeling experiments revealed that amiloride-type inhibitors bind to the interfacial domain of multiple core subunits (49 kDa, ND1, and PSST) and 39 kDa supernumerary subunit, although the latter does not make up the channel cavity in the current models. The binding of amilorides to the multiple target subunits was remarkably suppressed by other quinone-site inhibitors and SF-UQs. Taken together, the present results are difficult to reconcile with the current channel models. On the basis of comprehensive interpretations of the present results and of previous findings, we discuss the physiological relevance of these models.

Small heat shock proteins: Simplicity meets complexity [Cell Biology]

October 31st, 2018 by Martin Haslbeck, Sevil Weinkauf, Johannes Buchner

Small heat shock proteins (sHsps) are a ubiquitous and ancient family of ATP-independent molecular chaperones. A key characteristic of sHsps is that they exist in ensembles of iso-energetic oligomeric species differing in size. This property arises from a unique mode of assembly involving several parts of the subunits in a flexible manner. Current evidence suggests that smaller oligomers are more active chaperones. Thus, a shift in the equilibrium of the sHsp ensemble allows regulating the chaperone activity. Different mechanisms have been identified that reversibly change the oligomer equilibrium. The promiscuous interaction with non-native proteins generates complexes that can form aggregate-like structures from which native proteins are restored by ATP-dependent chaperones such as Hsp70 family members. In recent years, this basic paradigm has been expanded and new roles, new cofactors as well as variations in structure and regulation of sHsps have emerged.