Iron (Fe) deficiency is a major constraint for plant growth and affects the quality of edible plant parts. To investigate the mechanisms underlying Fe homeostasis in plants, Fe deficiency-induced changes in the protein profile of Arabidopsis (Arabidopsis thaliana) roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography-tandem mass spectrometry on a LTQ-Orbitrap with high-energy collision dissociation. A total of 4,454 proteins were identified with a false discovery rate of less than 1.1%, and 2,882 were reliably quantified.

Pyruvate kinase M2 isoform (PKM2) catalyzes the last step of glycolysis and plays an important role in tumor cell proliferation. Recent studies have reported that PKM2 also regulates apoptosis. However, the mechanisms underlying such a role of PKM2 remain elusive. Here we show that PKM2 translocates to mitochondria under oxidative stress. In the mitochondria, PKM2 interacts with and phosphorylates Bcl2 at threonine (T) 69. This phosphorylation prevents the binding of Cul3-based E3 ligase to Bcl2 and subsequent degradation of Bcl2. A chaperone protein, HSP90α1, is required for this function of PKM2. HSP90α1's ATPase activity launches a conformational change of PKM2 and facilitates interaction between PKM2 and Bcl2. Replacement of wild-type Bcl2 with phosphorylation-deficient Bcl2 T69A mutant sensitizes glioma cells to oxidative stress-induced apoptosis and impairs brain tumor formation in an orthotopic xenograft model. Notably, a peptide that is composed of the amino acid residues from 389 to 405 of PKM2, through which PKM2 binds to Bcl2, disrupts PKM2-Bcl2 interaction, promotes Bcl2 degradation and impairs brain tumor growth. In addition, levels of Bcl2 T69 phosphorylation, conformation-altered PKM2 and Bcl2 protein correlate with one another in specimens of human glioblastoma patients. Moreover, levels of Bcl2 T69 phosphorylation and conformation-altered PKM2 correlate with both grades and prognosis of glioma malignancy. Our findings uncover a novel mechanism through which mitochondrial PKM2 phosphorylates Bcl2 and inhibits apoptosis directly, highlight the essential role of PKM2 in ROS adaptation of cancer cells, and implicate HSP90-PKM2-Bcl2 axis as a potential target for therapeutic intervention in glioblastoma.

These results pinpoint GSDME-dependent pyroptosis as a previously unrecognized mechanism of action for molecular targeted agents to eradicate oncogene-addicted neoplastic cells, which may have important implications for the clinical development and optimal application of anticancer therapeutics.

BackgroundQuantitative information on gene activity at single cell-type resolution is essential for the understanding of how cells work and interact. Root hairs, or trichoblasts, tubular-shaped outgrowths of specialized cells in the epidermis, represent an ideal model for cell fate acquisition and differentiation in plants.ResultsHere, we provide an atlas of gene and protein expression in Arabidopsis root hair cells, generated by paired-end RNA sequencing and LC/MS-MS analysis of protoplasts from plants containing a pEXP7-GFP reporter construct. In total, transcripts of 23,034 genes were detected in root hairs. High-resolution proteome analysis led to the reliable identification of 2,447 proteins, 129 of which were differentially expressed between root hairs and non-root hair tissue. Dissection of pre-mRNA splicing patterns showed that all types of alternative splicing were cell type-dependent, and less complex in EXP7-expressing cells when compared to non-root hair cells. Intron retention was repressed in several transcripts functionally related to root hair morphogenesis, indicative of a cell type-specific control of gene expression by alternative splicing of pre-mRNA. Concordance between mRNA and protein expression was generally high, but in many cases mRNA expression was not predictive for protein abundance.ConclusionsThe integrated analysis shows that gene activity in root hairs is dictated by orchestrated, multilayered regulatory mechanisms that allow for a cell type-specific composition of functional components.

Macrophages are a dominant leukocyte population in the tumor microenvironment and actively promote cancer progression. However, the molecular mechanism underlying the role of macrophages remains poorly understood. Here we show that polarized M2 macrophages enhance 3-phosphoinositide-dependent protein kinase 1 (PDPK1)-mediated phosphoglycerate kinase 1 (PGK1) threonine (T) 243 phosphorylation in tumor cells by secreting interleukin-6 (IL-6). This phosphorylation facilitates a PGK1-catalyzed reaction toward glycolysis by altering substrate affinity. Inhibition of PGK1 T243 phosphorylation or PDPK1 in tumor cells or neutralization of macrophage-derived IL-6 abrogates macrophage-promoted glycolysis, proliferation, and tumorigenesis. In addition, PGK1 T243 phosphorylation correlates with PDPK1 activation, IL-6 expression, and macrophage infiltration in human glioblastoma multiforme (GBM). Moreover, PGK1 T243 phosphorylation also correlates with malignance and prognosis of human GBM. Our findings demonstrate a novel mechanism of macrophage-promoted tumor growth by regulating tumor cell metabolism, implicating the therapeutic potential to disrupt the connection between macrophages and tumor cells by inhibiting PGK1 phosphorylation.

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Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms.The removal of introns from the immature mRNA by a process called "pre-mRNA splicing" occurs in the vast majority of eukaryotic protein-coding genes. Pre-mRNA splicing is catalyzed by elaborate ribonucleoprotein megadalton complexes referred to as spliceosomes (Wahl et al., 2009). Multiple mRNA isoforms can be generated from a single gene locus by alternative splicing, potentially producing functionally distinct protein isoforms (Hsu and Hertel, 2009). In mammals, alternative splicing dramatically increases protein diversity, yielding proteins that differ in function, activity, binding properties, or subcellular localization, and is believed to account for the multiexonic gene expression diversity, generating an estimated 100,000 proteins encoded by 25,000 genes in humans (Modrek and Lee, 2002;Kelemen et al., 2013).In humans, more than 95% of the intron-containing genes are alternatively spliced (Pan et al., 2008). Alternative splicing is less prominent in plants, and its importance has been debated. Recent estimates based on RNA sequencing (RNA-seq) data sug...

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