Loss-of-function mutations of SQUINT (SQN)-which encodes theArabidopsis orthologue of cyclophilin 40 (CyP40)-cause the precocious expression of adult vegetative traits, an increase in carpel number, and produce abnormal spacing of flowers in the inflorescence. Here we show that the vegetative phenotype of sqn is attributable to the elevated expression of miR156-regulated members of the SPL family of transcription factors and provide evidence that this defect is a consequence of a reduction in the activity of ARGONAUTE1 (AGO1). Support for this latter conclusion was provided by the phenotypic similarity between hypomorphic alleles of AGO1 and null alleles of SQN and by the genetic interaction between sqn and these alleles. Our results suggest that AGO1, or an AGO1-interacting protein, is a major client of CyP40 and that miR156 and its targets play a central role in the regulation of vegetative phase change in Arabidopsis.CyP40 ͉ HSP90 ͉ miR156 ͉ phase change ͉ immunophilin S hoot growth in plants can be divided into juvenile, adult, and reproductive phases according to the character of the lateral organs (leaves and buds) produced during each phase. The juvenile-to-adult transition is known as vegetative phase change and is accompanied by changes in the shape and differentiation of leaves and by an increase in reproductive competence. Screens for mutations that accelerate vegetative phase change in Arabidopsis have produced a large number of genes, many of which encode proteins involved in the biogenesis or function of small RNAs that play key regulatory roles in this process. Although SQUINT (SQN) was one of the first vegetative phase change genes to be identified (1), the basis for its effect on this process remains unknown.SQN is an orthologue of the immunophilin cyclophilin 40 (Cyp40), a member of a large, evolutionarily conserved class of proteins that possess a peptidyl prolyl cis/trans isomerase (PPIase) domain. These proteins are commonly known as immunophilins because they were originally identified by virtue of their ability to bind the immunosuppressants cyclosporin A or FK506 (reviewed in ref.2). The 2 major families of immunophilins-cyclophilins and FK506-binding proteins (FKBPs)-have structurally distinct PPIase domains that are unrelated in amino acid sequence. Both families include low-molecular-weight proteins that consist solely of a PPIase domain, as well as larger proteins-such as CyP40-in which this domain is present in association with other functional domains. Arabidopsis possesses more than 50 immunophilins, most of which have no known function (3).The most intensively studied immunophilins in Arabidopsis are the multidomain FKBP proteins PASTICCINO1 (PAS1) and TWISTED DWARF (TWD)/ULTRACURVATA2 (UCU2). Like CyP40, these FKBP proteins possess an N-terminal PPIase domain and a C-terminal tetratricopeptide repeat (TPR) domain (4). Loss-of-function mutations in PAS1 produce small, distorted seedlings with defects in cytokinin and auxin signaling (5), whereas mutations in TWD/UCU2 produce plant...

The Orai channel is characterized by voltage independence, low conductance, and high Ca 2+ selectivity and plays an important role in Ca 2+ influx through the plasma membrane (PM). How the channel is activated and promotes Ca 2+ permeation is not well understood. Here, we report the crystal structure and cryo-electron microscopy (cryo-EM) reconstruction of a Drosophila melanogaster Orai (dOrai) mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which 6 transmembrane 1 (TM1) helices in the center form the ion-conducting pore, and 6 TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca 2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation, and Ca 2+ permeation.

No abstract

SETD3 is a member of the SET (Su(var)3–9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Previously, we identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin (Kwiatkowski et al., 2018). Here, we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses, supported by biochemical experiments and enzyme activity assays, indicate that the recognition and methylation of β-actin by SETD3 are highly sequence specific, and that both SETD3 and β-actin adopt pronounced conformational changes upon binding to each other. In conclusion, this study is the first to show a catalytic mechanism of SETD3-mediated histidine methylation on β-actin, which not only throws light on the protein histidine methylation phenomenon but also facilitates the design of small molecule inhibitors of SETD3.

Overexpressed Aurora-A kinase promotes tumor growth through various pathways, but whether Aurora-A is also involved in metabolic reprogramming-mediated cancer progression remains unknown. Here, we report that Aurora-A directly interacts with and phosphorylates lactate dehydrogenase B (LDHB), a subunit of the tetrameric enzyme LDH that catalyzes the interconversion between pyruvate and lactate. Aurora-A-mediated phosphorylation of LDHB serine 162 significantly increases its activity in reducing pyruvate to lactate, which efficiently promotes NAD+ regeneration, glycolytic flux, lactate production and bio-synthesis with glycolytic intermediates. Mechanistically, LDHB serine 162 phosphorylation relieves its substrate inhibition effect by pyruvate, resulting in remarkable elevation in the conversions of pyruvate and NADH to lactate and NAD+. Blocking S162 phosphorylation by expression of a LDHB-S162A mutant inhibited glycolysis and tumor growth in cancer cells and xenograft models. This study uncovers a function of Aurora-A in glycolytic modulation and a mechanism through which LDHB directly contributes to the Warburg effect.

Store-operated calcium entry (SOCE) is a major pathway for calcium ions influx into cells and has a critical role in various cell functions. Here we demonstrate that calcium-bound calmodulin (Ca2+-CaM) binds to the core region of activated STIM1. This interaction facilitates slow Ca2+-dependent inactivation after Orai1 channel activation by wild-type STIM1 or a constitutively active STIM1 mutant. We define the CaM-binding site in STIM1, which is adjacent to the STIM1–Orai1 coupling region. The binding of Ca2+-CaM to activated STIM1 disrupts the STIM1–Orai1 complex and also disassembles STIM1 oligomer. Based on these results we propose a model for the calcium-bound CaM-regulated deactivation of SOCE.

31SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain 32 protein superfamily and plays important roles in hypoxic pulmonary hypertension, 33 muscle differentiation, and carcinogenesis. In a previous paper (Kwiatkowski et al. 34 2018), we have identified SETD3 as the actin-specific methyltransferase that 35 methylates the N 3 of His73 on β-actin. Here we present two structures of 36 S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified 37 β-actin peptide or its His-methylated variant. Structural analyses supported by the 38 site-directed mutagenesis experiments and the enzyme activity assays indicated that 39 the recognition and methylation of β-actin by SETD3 is highly sequence specific, and 40 both SETD3 and β-actin adopt pronounce conformational changes upon binding to 41 each other. In conclusion, the structural research uncovers the molecular mechanism 42 of sequence-selective histidine methylation by SETD3, which not only throws light on 43 protein histidine methylation phenomenon, but also facilitates the design of small 44 molecule inhibitors of SETD3. 45 46 91 by SETD3. 92 93With the two solved β-actin peptide-SETD3 structures, we uncover that SETD3 94 recognizes a fragment of β-actin in a sequence-dependent manner and utilizes a 95 specific pocket to catalyze the N 3 -methylation of His73. Moreover, a comprehensive 96 structural, biochemical and enzymatic profiling of SETD3 allows us to pinpoint its 97 key residues important for substrate recognition and subsequent methylation. 98Therefore, the structural research, supplemented by biochemical and enzymatic 99 experiments, not only provides insights into the catalytic mechanism of SETD3, but 100 also will facilitate the design of specific inhibitors of SETD3 enzyme. 101 102 RESULTS 103SETD3 binds to and methylates β-actin 104 Since SETD3 was identified as a histidine methyltransferase that methylates His73 of 105 β-actin (Kwiatkowski et al., 2018, Wilkinson et al., 2019, we purified the core region 106 of SETD3 (aa 2-502) and studied by ITC its binding to a His73-containing fragment 107 of β-actin (aa 66-88) ( Figures 1A). The ITC binding experiment showed that SETD3 108 bound to the β-actin peptide with a Kd of 0.17 μM ( Figure 1B and Table 1). Given 109 that SETD3 was also reported to be a putative lysine methyltransferase that 110 methylates Lys4 and Lys36 of histone H3 (Eom et al., 2011), we also verified the 111 binding of SETD3 to two different histone peptides, H3K4(1-23) and H3K36(25-47), 112 and found that neither of them binds to SETD3 (Table 1).113 114 Furthermore, we tested the activity of SETD3 on β-actin(66-88), H3K4(1-23), and 115 H3K36(25-47) by mass spectrometry, and we found that SETD3 methylates β-actin 116 peptide (Figure 1-figure supplement 1A), but does not modify either H3K4 or H3K36 117 (Figure 1-figure supplement 1B-1C). No methylated product was detected for any of 118 above peptides in the presence of AdoMet without the addition of SETD3 (Figure 119 1-figure supple...

Hec1 (Highly Expressed in Cancer 1) or Nek2 (NIMA-related kinase 2) is often overexpressed in cancers with poor prognosis. Both are critical mitotic regulators and phosphorylation of Hec1 S165 by Nek2 is required for proper chromosome segregation. Therefore, inactivation of Hec1 and Nek2 by targeting their interaction with small molecules represents an ideal strategy for tackling these types of cancers. Here, we showed that new derivatives of INH (Inhibitor for Nek2 and Hec1 binding) bind to Hec1 at amino acids 394–408 on W395, L399 and K400 residues, effectively blocking Hec1 phosphorylation on S165 by Nek2, and killing cancer cells at the nanomolar range. Mechanistically, the D-box (destruction-box) region of Nek2 specifically binds to Hec1 at amino acids 408–422, immediately adjacent to the INH binding motif. Subsequent binding of Nek2 to INH-bound Hec1 triggered proteasome-mediated Nek2 degradation, whereas the Hec1 binding defective Nek2 mutant, Nek2 R361L, resisted INH-induced Nek2 degradation. This finding unveils a novel drug-action mechanism where the binding of INHs to Hec1 forms a virtual death-trap to trigger Nek2 degradation and eventually cell death. Furthermore, analysis of the gene expression profiles of breast cancer patient samples revealed that co-elevated expressions of Hec1 and Nek2 correlated with the shortest survival. Treatment of mice with this kind of tumor with INHs significantly suppressed tumor growth without obvious toxicity. Taken together, the new INH derivatives are suitable for translation into clinical application.