Structural basis and regulation of the reductive stress response

The integrated stress response (ISR) tunes the rate of protein synthesis. Control is exerted by phosphorylation of the general translation initiation factor eIF2. eIF2 is a GTPase, that becomes activated by eIF2B, a large two-fold symmetric and heterodecameric complex that functions as eIF2's dedicated nucleotide exchange factor. Phosphorylation converts eIF2 from substrate into an inhibitor of eIF2B. We report cryoEM structures of eIF2 bound to eIF2B in the dephosphorylated state. The structures reveal that the eIF2B decamer is a static platform upon which one or two flexible eIF2 trimers bind and align with eIF2B's catalytic centers to catalyze guanine nucleotide exchange. Phosphorylation refolds eIF2, allowing it to contact eIF2B at a different interface and, we surmise, thereby sequesters it into a non-productive complex.

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Molecular and Structural Basis of Cross-Reactivity in M. tuberculosis Toxin–Antitoxin Systems

Toxins ◽

10.3390/toxins12080481 ◽

2020 ◽

Vol 12 (8) ◽

pp. 481 ◽

Cited By ~ 1

Author(s):

Himani Tandon ◽

Akhila Melarkode Vattekatte ◽

Narayanaswamy Srinivasan ◽

Sankaran Sandhya

Keyword(s):

Mycobacterium Tuberculosis ◽

Stress Response ◽

Structural Analysis ◽

Chemical Nature ◽

Structural Models ◽

Cross Reactivity ◽

Structural Basis ◽

Detailed Structural Analysis ◽

Response Network ◽

Computational Analyses

Mycobacterium tuberculosis genome encodes over 80 toxin–antitoxin (TA) systems. While each toxin interacts with its cognate antitoxin, the abundance of TA systems presents an opportunity for potential non-cognate interactions. TA systems mediate manifold interactions to manage pathogenicity and stress response network of the cell and non-cognate interactions may play vital roles as well. To address if non-cognate and heterologous interactions are feasible and to understand the structural basis of their interactions, we have performed comprehensive computational analyses on the available 3D structures and generated structural models of paralogous M. tuberculosis VapBC and MazEF TA systems. For a majority of the TA systems, we show that non-cognate toxin–antitoxin interactions are structurally incompatible except for complexes like VapBC15 and VapBC11, which show similar interfaces and potential for cross-reactivity. For TA systems which have been experimentally shown earlier to disfavor non-cognate interactions, we demonstrate that they are structurally and stereo-chemically incompatible. For selected TA systems, our detailed structural analysis identifies specificity conferring residues. Thus, our work improves the current understanding of TA interfaces and generates a hypothesis based on congenial binding site, geometric complementarity, and chemical nature of interfaces. Overall, our work offers a structure-based explanation for non-cognate toxin-antitoxin interactions in M. tuberculosis.

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Structural basis for eIF2B inhibition in integrated stress response

Science ◽

10.1126/science.aaw4104 ◽

2019 ◽

Vol 364 (6439) ◽

pp. 495-499 ◽

Cited By ~ 23

Author(s):

Kazuhiro Kashiwagi ◽

Takeshi Yokoyama ◽

Madoka Nishimoto ◽

Mari Takahashi ◽

Ayako Sakamoto ◽

...

Keyword(s):

Stress Response ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Structural Basis ◽

Dependent Manner ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Electron Microscopic ◽

Eukaryotic Translation ◽

Initiation Factor 2

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5′-triphosphate–dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo–electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.

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Structural Basis for eIF2B Inhibition in Integrated Stress Response

10.1101/503540 ◽

2018 ◽

Author(s):

Kazuhiro Kashiwagi ◽

Takeshi Yokoyama ◽

Madoka Nishimoto ◽

Mari Takahashi ◽

Ayako Sakamoto ◽

...

Keyword(s):

Stress Response ◽

Translational Control ◽

Binding Mode ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Structural Basis ◽

Dependent Manner ◽

Integrated Stress Response ◽

Nucleotide Exchange ◽

Electron Microscopic

AbstractA core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers methionylated initiator tRNA to the ribosome in a GTP-dependent manner. In contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor eIF2B, which leads to a deficiency of active eIF2 and resultant global translation repression. To unveil the mechanism by which the eIF2 phosphorylation status regulates the eIF2B nucleotide exchange activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. Intriguingly, the unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active “productive” and nucleotide exchange-inactive “nonproductive” modes, respectively. The nonproductive-mode phosphorylated eIF2, extending from one of the two eIF2B “central cavities”, not only prevents nucleotide exchange on itself, but also sterically prevents unphosphorylated eIF2 from productively binding on the other central cavity of eIF2B, which explains how phosphorylated eIF2 inhibits eIF2B.One Sentence SummaryA drastic change in the binding mode of eIF2 to eIF2B induces translational control in stress.

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Genome-wide analysis of the apple CaCA superfamily reveals that MdCAX proteins are involved in the abiotic stress response as calcium transporters

BMC Plant Biology ◽

10.1186/s12870-021-02866-1 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Ke Mao ◽

Jie Yang ◽

Min Wang ◽

Huayu Liu ◽

Xin Guo ◽

...

Keyword(s):

Abiotic Stress ◽

Stress Response ◽

Plant Species ◽

Stress Responses ◽

Calcium Transport ◽

Sequence Similarity ◽

Structural Basis ◽

Functional Study ◽

Transport Capacity ◽

Abiotic Stress Response

Abstract Background Calcium (Ca2+) plays an important role in plant growth and development, and the maintenance of calcium homeostasis is necessary for the survival of all plant species. Ca2+/H+ exchangers (CAXs) are a subgroup of the CaCA (Ca2+/cation antiporter) superfamily. In general, CAX proteins mediate cytosolic Ca2+ entry into vacuoles to prevent excessive accumulation of Ca2+ in the cytosol. The CaCA superfamily has been identified and characterised in many plant species; however, characterisation of the CaCA superfamily and functional study of apple CAX proteins have yet to be conducted in apple (Malus × domestica Borkh.). Results Here, we identified 21 CaCA family proteins in apple for the first time. Phylogenetic and gene structure analysis, as well as prediction of conserved motifs, suggested that these proteins could be classified into four groups: CAX, CCX, NCL, and MHX. Expression analysis showed that the 10 MdCAX genes we cloned strongly responded to calcium and abiotic stress treatments. Collinearity analysis and characterisation of calcium transport capacity resulted in the identification of a pair of segmental duplication genes: MdCAX3L-1 and MdCAX3L-2; MdCAX3L-2 showed strong calcium transport capacity, whereas MdCAX3L-1 showed no calcium transport capacity. Yeast two-hybrid (Y2H) assays showed that these two proteins could interact with each other. The high sequence similarity (94.6%) makes them a good model for studying the crucial residues and structural basis of the calcium transport of CAX proteins. Prediction of the protein interaction network revealed several proteins that may interact with CAX proteins and play important roles in plant stress responses, such as SOS2, CXIP1, MHX, NRAMP3, and MTP8. Conclusions Our analysis indicated that MdCAX proteins have strong calcium transport capacity and are involved in the abiotic stress response in apple. These findings provide new insight and rich resources for future studies of MdCAX proteins in apple.

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Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1117003109 ◽

2012 ◽

Vol 109 (21) ◽

pp. E1405-E1414 ◽

Cited By ~ 42

Author(s):

S. Campagne ◽

F. F. Damberger ◽

A. Kaczmarczyk ◽

A. Francez-Charlot ◽

F. H.- T. Allain ◽

...

Keyword(s):

Stress Response ◽

Sigma Factor ◽

Structural Basis ◽

General Stress Response ◽

General Stress

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Organization of tight junctions in the choroid plexus epithelium

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082546 ◽

1978 ◽

Vol 36 (2) ◽

pp. 336-337

Author(s):

B. Van Deurs ◽

J. K. Koehler

Keyword(s):

Choroid Plexus ◽

Present Report ◽

Freeze Fracture ◽

Barrier Properties ◽

Cell Junctions ◽

Structural Basis ◽

Apical Surface ◽

Choroid Plexus Epithelium ◽

Solid Nitrogen ◽

Special Composition

The choroid plexus epithelium constitutes a blood-cerebrospinal fluid (CSF) barrier, and is involved in regulation of the special composition of the CSF. The epithelium is provided with an ouabain-sensitive Na/K-pump located at the apical surface, actively pumping ions into the CSF. The choroid plexus epithelium has been described as “leaky” with a low transepithelial resistance, and a passive transepithelial flux following a paracellular route (intercellular spaces and cell junctions) also takes place. The present report describes the structural basis for these “barrier” properties of the choroid plexus epithelium as revealed by freeze fracture.Choroid plexus from the lateral, third and fourth ventricles of rats were used. The tissue was fixed in glutaraldehyde and stored in 30% glycerol. Freezing was performed either in liquid nitrogen-cooled Freon 22, or directly in a mixture of liquid and solid nitrogen prepared in a special vacuum chamber. The latter method was always used, and considered necessary, when preparations of complementary (double) replicas were made.

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