Tackling Salinity in Sustainable Agriculture—What Developing Countries May Learn from Approaches of the Developed World

Soil salinity is a common problem of the developing world as well as the developed world. However, the pace to reduce salinity is much slower in the developing world. The application of short-term approaches with an unsustainable supply of funds are the major reasons of low success. In contrast, the developed world has focused on long-term and sustainable techniques, and considerable funds per unit area have been allocated to reduce soil salinity. Here, we review the existing approaches in both worlds. Approaches like engineering and nutrient use were proven to be unsustainable, while limited breeding and biosaline approaches had little success in the developing countries. In contrast, advanced breeding and genetics tools were implemented in the developed countries to improve the salinity tolerance of different crops with more success. Resultantly, developed countries not only reduced the area for soil salinity at a higher rate, but more sustainable and cheaper ways to resolve the issue were implemented at the farmers’ field. Similarly, plant microbial approaches and the application of fertigation through drip irrigation have great potential for both worlds, and farmer participatory approaches are required to obtain fruitful outcomes. In this regard, a challenging issue is the transition of sustainable approaches from developed countries to developing ones, and possible methods for this are discussed

The p110 delta structure: mechanisms for selectivity and potency of new PI(3)K inhibitors.

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies

Identification of Novel ERK2 substrates through use of an engineered kinase and ATP analogs

The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified bydifferential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade

Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new ATP-competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and PIK3CA mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, while those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor 4E-BP1, but not ribosomal protein S6. In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242 induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared to KRAS WT controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1

p27 allosterically activates cyclin-dependent kinase 4 and antagonizes palbociclib inhibition.

The p27 protein is a canonical negative regulator of cell proliferation and acts primarily by inhibiting cyclin-dependent kinases (CDKs). Under some circumstances, p27 is associated with active CDK4, but no mechanism for activation has been described. We found that p27, when phosphorylated by tyrosine kinases, allosterically activated CDK4 in complex with cyclin D1 (CDK4-CycD1). Structural and biochemical data revealed that binding of phosphorylated p27 (phosp27) to CDK4 altered the kinase adenosine triphosphate site to promote phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and other substrates. Surprisingly, purified and endogenous phosp27-CDK4-CycD1 complexes were insensitive to the CDK4-targeting drug palbociclib. Palbociclib instead primarily targeted monomeric CDK4 and CDK6 (CDK4/6) in breast tumor cells. Our data characterize phosp27-CDK4-CycD1 as an active Rb kinase that is refractory to clinically relevant CDK4/6 inhibitors

Structures of PI4KIIIβ complexes show simultaneous recruitment of Rab11 and its effectors

Phosphatidylinositol 4-kinases (PI4Ks) and small guanosine triphosphatases (GTPases) are essential for processes that require expansion and remodeling of phosphatidylinositol 4-phosphate (PI4P)-containing membranes, including cytokinesis, intracellular development of malarial pathogens, and replication of a wide range of RNA viruses. However, the structural basis for coordination of PI4K, GTPases and their effectors is unknown. Here, we describe structures of PI4KB (PI4KIIIβ) bound to the small GTPase Rab11a without and with the Rab11 effector protein FIP3. The Rab11-PI4KIIIβ interface is unique compared with known structures of Rab complexes, and does not involve switch regions used by GTPase effectors. Our data provide a mechanism for how PI4KIIIβ coordinates Rab11 and its effectors on PI4P-enriched membranes, and also provide strategies for the design of specific inhibitors that could potentially target plasmodial PI4KIIIβ to combat malaria

Evolution of enhanced innate immune evasion by SARS-CoV-2

Emergence of SARS-CoV-2 variants of concern (VOCs) suggests viral adaptation to enhance human-to-human transmission1,2. Although much effort has focused on characterisation of spike changes in VOCs, mutations outside spike likely contribute to adaptation. Here we used unbiased abundance proteomics, phosphoproteomics, RNAseq and viral replication assays to show that isolates of the Alpha (B.1.1.7) variant3 more effectively suppress innate immune responses in airway epithelial cells, compared to first wave isolates. We found that Alpha has dramatically increased subgenomic RNA and protein levels of N, Orf9b and Orf6, all known innate immune antagonists. Expression of Orf9b alone suppressed the innate immune response through interaction with TOM70, a mitochondrial protein required for RNA sensing adaptor MAVS activation. Moreover, the activity of Orf9b and its association with TOM70 was regulated by phosphorylation. We propose that more effective innate immune suppression, through enhanced expression of specific viral antagonist proteins, increases the likelihood of successful Alpha transmission, and may increase in vivo replication and duration of infection4. The importance of mutations outside Spike in adaptation of SARS-CoV-2 to humans is underscored by the observation that similar mutations exist in the Delta and Omicron N/Orf9b regulatory regions

Harnessing genetic potential of wheat germplasm banks through impact-oriented-prebreeding for future food and nutritional security

The value of exotic wheat genetic resources for accelerating grain yield gains is largely unproven and unrealized. We used next-generation sequencing, together with multi-environment phenotyping, to study the contribution of exotic genomes to 984 three-way-cross-derived (exotic/elite1//elite2) pre-breeding lines (PBLs). Genomic characterization of these lines with haplotype map-based and SNP marker approaches revealed exotic specific imprints of 16.1 to 25.1%, which compares to theoretical expectation of 25%. A rare and favorable haplotype (GT) with 0.4% frequency in gene bank identified on chromosome 6D minimized grain yield (GY) loss under heat stress without GY penalty under irrigated conditions. More specifically, the ‘T’ allele of the haplotype GT originated in Aegilops tauschii and was absent in all elite lines used in study. In silico analysis of the SNP showed hits with a candidate gene coding for isoflavone reductase IRL-like protein in Ae. tauschii. Rare haplotypes were also identified on chromosomes 1A, 6A and 2B effective against abiotic/biotic stresses. Results demonstrate positive contributions of exotic germplasm to PBLs derived from crosses of exotics with CIMMYT’s best elite lines. This is a major impact-oriented pre-breeding effort at CIMMYT, resulting in large-scale development of PBLs for deployment in breeding programs addressing food security under climate change scenarios

Structural and functional basis for RNA cleavage by Ire1

BACKGROUND: The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. RESULTS: This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing \u3e/=7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. CONCLUSIONS: Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L