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Strigolactones (SLs) are a new class of phytohormones that also act as germination stimulants for root parasitic plants, such as Striga spp., and as branching factors for symbiotic arbuscular mycorrhizal fungi. Sources for natural SLs are very limited. Hence, efficient and simple SL analogs are needed for elucidating SL-related biological processes as well as for agricultural applications. Based on the structure of the non-canonical SL methyl carlactonoate, we developed a new, easy to synthesize series of analogs, termed methyl phenlactonoates (MPs), evaluated their efficacy in exerting different SL functions, and determined their affinity for SL receptors from rice and Striga hermonthica. Most of the MPs showed considerable activity in regulating plant architecture, triggering leaf senescence, and inducing parasitic seed germination. Moreover, some MPs outperformed GR24, a widely used SL analog with a complex structure, in exerting particular SL functions, such as modulating Arabidopsis roots architecture and inhibiting rice tillering. Thus, MPs will help in elucidating the functions of SLs and are promising candidates for agricultural applications. Moreover, MPs demonstrate that slight structural modifications clearly impact the efficiency in exerting particular SL functions, indicating that structural diversity of natural SLs may mirror a functional specificity.

This corrects the article DOI: 10.1038/gim.2017.22.

Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii , a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinear hox gene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation of hox -controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient.

Because only 0.01% of prokaryotic genospecies can be cultured and in situ observations are often impracticable, culture-independent methods are required to understand microbial life and harness potential applications of microbes. Here, we report a methodology for the production of proteins with desired functions based on single amplified genomes (SAGs) from unculturable species. We use this method to resurrect an alcohol dehydrogenase (ADH/D1) from an uncharacterized halo-thermophilic archaeon collected from a brine pool at the bottom of the Red Sea. Our crystal structure of 5,6-dihydroxy NADPH-bound ADH/D1 combined with biochemical analyses reveal the molecular features of its halo-thermophily, its unique habitat adaptations, and its possible reaction mechanism for atypical oxygen activation. Our strategy offers a general guide for using SAGs as a source for scientific and industrial investigations of "microbial dark matter."

Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Variant nomenclature discrepancy was identified in the article.

Correction: After publication of the article [1], it has been brought to our attention that there is a nomenclature issue with this article. At the time of acceptance, the VARS2 mutation was considered equivalent to the VARS2 mutation. However, this has changed so that VARS now only refers to shorter mitochondrial sequence of valyl-tRNA synthesase containing 1093 amino acids. "Therefore, in the context of this article, every usage of "VARS2" should be replaced with "VARS" when referring to the causative variant".

Background: Most mitochondrial and cytoplasmic aminoacyl-tRNA synthetases (aaRSs) are encoded by nuclear genes. Syndromic disorders resulting from mutation of aaRSs genes display significant phenotypic heterogeneity. We expand aaRSs-related phenotypes through characterization of the clinical and molecular basis of a novel autosomal-recessive syndrome manifesting severe mental retardation, ataxia, speech impairment, epilepsy, short stature, microcephaly, hypogonadism, and growth hormone deficiency., Results: A G>A variant in exon 29 of VARS2 (c.3650G>A) (NM_006295) was identified in the index case. This homozygous variant was confirmed by Sanger sequencing and segregated with disease in the family studied. The c.3650G>A change results in alteration of arginine to histidine at residue 1217 (R1217H) of the mature protein and is predicted to be pathogenic., Conclusions: These findings contribute to a growing list of aaRSs disorders, broadens the spectrum of phenotypes attributable to VARS2 mutations, and provides new insight into genotype-phenotype correlations among the mitochondrial synthetase genes.

Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of neurodevelopmental disorders with poor prognosis. Recent discoveries have greatly expanded the repertoire of genes that are mutated in epileptic encephalopathies and DEE, often in a de novo fashion, but in many patients, the disease remains molecularly uncharacterized. Here, we describe a new form of DEE in patients with likely deleterious biallelic variants in PTPN23. The phenotype is characterized by early onset drug-resistant epilepsy, severe and global developmental delay, microcephaly, and sometimes premature death. PTPN23 encodes a tyrosine phosphatase with strong brain expression, and its knockout in mouse is embryonically lethal. Structural modeling supports a deleterious effect of the identified alleles. Our data suggest that PTPN23 mutations cause a rare severe form of autosomal-recessive DEE in humans, a finding that requires confirmation.

Intellectual disability (ID) is a common morbid condition with a wide range of etiologies. The list of monogenic forms of ID has increased rapidly in recent years thanks to the implementation of genomic sequencing techniques. In this study, we describe the phenotypic and genetic findings of 68 families (105 patients) all with novel ID-related variants. In addition to established ID genes, including ones for which we describe unusual mutational mechanism, some of these variants represent the first confirmatory disease-gene links following previous reports (TRAK1, GTF3C3, SPTBN4 and NKX6-2), some of which were based on single families. Furthermore, we describe novel variants in 14 genes that we propose as novel candidates (ANKHD1, ASTN2, ATP13A1, FMO4, MADD, MFSD11, NCKAP1, NFASC, PCDHGA10, PPP1R21, SLC12A2, SLK, STK32C and ZFAT). We highlight MADD and PCDHGA10 as particularly compelling candidates in which we identified biallelic likely deleterious variants in two independent ID families each. We also highlight NCKAP1 as another compelling candidate in a large family with autosomal dominant mild intellectual disability that fully segregates with a heterozygous truncating variant. The candidacy of NCKAP1 is further supported by its biological function, and our demonstration of relevant expression in human brain. Our study expands the locus and allelic heterogeneity of ID and demonstrates the power of positional mapping to reveal unusual mutational mechanisms.

Infertility is a relatively common disorder of the reproductive system and remains unexplained in many cases. In vitro fertilization techniques have uncovered previously unrecognized infertility phenotypes, including oocyte maturation arrest, the molecular etiology of which remains largely unknown. We report two families affected by female-limited infertility caused by oocyte maturation failure. Positional mapping and whole-exome sequencing revealed two homozygous, likely deleterious variants in PATL2, each of which fully segregates with the phenotype within the respective family. PATL2 encodes a highly conserved oocyte-specific mRNP repressor of translation. Previous data have shown the strict requirement for PATL2 in oocyte-maturation in model organisms. Data gathered from the families in this study suggest that the role of PATL2 is conserved in humans and expand our knowledge of the factors that are necessary for female meiosis., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.

Purpose: The purpose of this study is to describe recessive alleles in strictly dominant genes. Identifying recessive mutations in genes for which only dominant disease or risk alleles have been reported can expand our understanding of the medical relevance of these genes both phenotypically and mechanistically. The Saudi population is enriched for autozygosity, which enhances the homozygous occurrence of alleles, including pathogenic alleles in genes that have been associated only with a dominant inheritance pattern., Methods: Exome sequencing of patients from consanguineous families with likely recessive phenotypes was performed. In one family, the genotype of the deceased children was inferred from their parents due to lack of available samples., Results: We describe the identification of 11 recessive variants (5 of which are reported here for the first time) in 11 genes for which only dominant disease or risk alleles have been reported. The observed phenotypes for these recessive variants were novel (e.g., FBN2-related myopathy and CSF1R-related brain malformation and osteopetrosis), typical (e.g., ACTG2-related visceral myopathy), or an apparently healthy state (e.g., PDE11A), consistent with the corresponding mouse knockout phenotypes., Conclusion: Our results show that, in the era of genomic sequencing and "reverse phenotyping," recessive variants in dominant genes should not be dismissed based on perceived "incompatibility" with the patient's phenotype before careful consideration.Genet Med advance online publication 06 April 2017.

This corrects the article DOI: 10.1038/nature21370.

Intellectual disability (ID) is a measurable phenotypic consequence of genetic and environmental factors. In this study, we prospectively assessed the diagnostic yield of genomic tools (molecular karyotyping, multi-gene panel and exome sequencing) in a cohort of 337 ID subjects as a first-tier test and compared it with a standard clinical evaluation performed in parallel. Standard clinical evaluation suggested a diagnosis in 16% of cases (54/337) but only 70% of these (38/54) were subsequently confirmed. On the other hand, the genomic approach revealed a likely diagnosis in 58% (n=196). These included copy number variants in 14% (n=54, 15% are novel), and point mutations revealed by multi-gene panel and exome sequencing in the remaining 43% (1% were found to have Fragile-X). The identified point mutations were mostly recessive (n=117, 81%), consistent with the high consanguinity of the study cohort, but also X-linked (n=8, 6%) and de novo dominant (n=19, 13%). When applied directly on all cases with negative molecular karyotyping, the diagnostic yield of exome sequencing was 60% (77/129). Exome sequencing also identified likely pathogenic variants in three novel candidate genes (DENND5A, NEMF and DNHD1) each of which harbored independent homozygous mutations in patients with overlapping phenotypes. In addition, exome sequencing revealed de novo and recessive variants in 32 genes (MAMDC2, TUBAL3, CPNE6, KLHL24, USP2, PIP5K1A, UBE4A, TP53TG5, ATOH1, C16ORF90, SLC39A14, TRERF1, RGL1, CDH11, SYDE2, HIRA, FEZF2, PROCA1, PIANP, PLK2, QRFPR, AP3B2, NUDT2, UFC1, BTN3A2, TADA1, ARFGEF3, FAM160B1, ZMYM5, SLC45A1, ARHGAP33 and CAPS2), which we highlight as potential candidates on the basis of several lines of evidence, and one of these genes (SLC39A14) was biallelically inactivated in a potentially treatable form of hypermanganesemia and neurodegeneration. Finally, likely causal variants in previously published candidate genes were identified (ASTN1, HELZ, THOC6, WDR45B, ADRA2B and CLIP1), thus supporting their involvement in ID pathogenesis. Our results expand the morbid genome of ID and support the adoption of genomics as a first-tier test for individuals with ID.

Chenopodium quinoa (quinoa) is a highly nutritious grain identified as an important crop to improve world food security. Unfortunately, few resources are available to facilitate its genetic improvement. Here we report the assembly of a high-quality, chromosome-scale reference genome sequence for quinoa, which was produced using single-molecule real-time sequencing in combination with optical, chromosome-contact and genetic maps. We also report the sequencing of two diploids from the ancestral gene pools of quinoa, which enables the identification of sub-genomes in quinoa, and reduced-coverage genome sequences for 22 other samples of the allotetraploid goosefoot complex. The genome sequence facilitated the identification of the transcription factor likely to control the production of anti-nutritional triterpenoid saponins found in quinoa seeds, including a mutation that appears to cause alternative splicing and a premature stop codon in sweet quinoa strains. These genomic resources are an important first step towards the genetic improvement of quinoa.

Background: Finding a source from which high-energy-density biofuels can be derived at an industrial scale has become an urgent challenge for renewable energy production. Some microorganisms can produce free fatty acids (FFA) as precursors towards such high-energy-density biofuels. In particular, photosynthetic cyanobacteria are capable of directly converting carbon dioxide into FFA. However, current engineered strains need several rounds of engineering to reach the level of production of FFA to be commercially viable; thus new chassis strains that require less engineering are needed. Although more than 120 cyanobacterial genomes are sequenced, the natural potential of these strains for FFA production and excretion has not been systematically estimated., Results: Here we present the FFA SC (FFASC), an in silico screening method that evaluates the potential for FFA production and excretion of cyanobacterial strains based on their proteomes. A literature search allowed for the compilation of 64 proteins, most of which influence FFA production and a few of which affect FFA excretion. The proteins are classified into 49 orthologous groups (OGs) that helped create rules used in the scoring/ranking of algorithms developed to estimate the potential for FFA production and excretion of an organism. Among 125 cyanobacterial strains, FFASC identified 20 candidate chassis strains that rank in their FFA producing and excreting potential above the specifically engineered reference strain, Synechococcus sp. PCC 7002. We further show that the top ranked cyanobacterial strains are unicellular and primarily include Prochlorococcus (order Prochlorales) and marine Synechococcus (order Chroococcales) that cluster phylogenetically. Moreover, two principal categories of enzymes were shown to influence FFA production the most: those ensuring precursor availability for the biosynthesis of lipids, and those involved in handling the oxidative stress associated to FFA synthesis., Conclusion: To our knowledge FFASC is the first in silico method to screen cyanobacteria proteomes for their potential to produce and excrete FFA, as well as the first attempt to parameterize the criteria derived from genetic characteristics that are favorable/non-favorable for this purpose. Thus, FFASC helps focus experimental evaluation only on the most promising cyanobacteria.

Ectodermal dysplasia is a highly heterogeneous group of disorders that variably affect the derivatives of the ectoderm, primarily skin, hair, nails and teeth. TP63, itself mutated in ectodermal dysplasia, links many other ectodermal dysplasia disease genes through a regulatory network that maintains the balance between proliferation and differentiation of the epidermis and other ectodermal derivatives. The ectodermal knockout phenotype of five mouse genes that regulate and/or are regulated by TP63 (Irf6, Ikkα, Ripk4, Stratifin, and Kdf1) is strikingly similar and involves abnormal balance towards proliferation at the expense of differentiation, but only the first three have corresponding ectodermal phenotypes in humans. We describe a multigenerational Saudi family with an autosomal dominant form of hypohidrotic ectodermal dysplasia in which positional mapping and exome sequencing identified a novel variant in KDF1 that fully segregates with the phenotype. The recapitulation of the phenotype we observe in this family by the Kdf1-/- mouse suggests a causal role played by the KDF1 variant.

Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver ( ContaMiner ) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for 'crystallization and preliminary X-ray analysis' publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data.

Glycine cleavage system (GCS) catalyzes the degradation of glycine and disruption of its components encoded by GLDC, AMT and GCSH are the only known causes of glycine encephalopathy, also known as non-ketotic hyperglycinemia (NKH). In this report, we describe a consanguineous family with one child who presented with NKH, but harbored no pathogenic variants in any of the three genes linked to this condition. Whole-exome sequencing revealed a novel homozygous missense variant in exon 9 of SLC6A9 NM_201649.3: c.1219 A>G (p.Ser407Gly) that segregates with the disease within the family. This variant replaces the highly conserved S407 in the ion-binding site of this glycine transporter and is predicted to disrupt its function. In murine model, knockout of Slc6a9 is associated with equivalent phenotype of NKH, namely respiratory distress and hypotonia. This is the first demonstration that mutation of the glycine transporter can be associated with NKH in humans., Competing Interests: The authors declare that they have no competing interests. Funding statement This research received no funding grant from public, commercial or not-for-profit sectors.

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

To better understand how the apoptosis repressor with caspase recruitment domain (ARC) protein confers drug resistance in acute myeloid leukemia (AML), we investigated the role of ARC in regulating leukemia-mesenchymal stromal cell (MSC) interactions. In addition to the previously reported effect on AML apoptosis, we have demonstrated that ARC enhances migration and adhesion of leukemia cells to MSCs both in vitro and in a novel human extramedullary bone/bone marrow mouse model. Mechanistic studies revealed that ARC induces IL1β expression in AML cells and increases CCL2, CCL4, and CXCL12 expression in MSCs, both through ARC-mediated activation of NFκB. Expression of these chemokines in MSCs increased by AML cells in an ARC/IL1β-dependent manner; likewise, IL1β expression was elevated when leukemia cells were co-cultured with MSCs. Further, cells from AML patients expressed the receptors for and migrated toward CCL2, CCL4, and CXCL12. Inhibition of IL1β suppressed AML cell migration and sensitized the cells co-cultured with MSCs to chemotherapy. Our results suggest the existence of a complex ARC-regulated circuit that maintains intimate connection of AML with the tumor microenvironment through NFκB/IL1β-regulated chemokine receptor/ligand axes and reciprocal crosstalk resulting in cytoprotection. The data implicate ARC as a promising drug target to potentially sensitize AML cells to chemotherapy.

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Motivation: The inherent promiscuity of small molecules towards protein targets impedes our understanding of healthy versus diseased metabolism. This promiscuity also poses a challenge for the pharmaceutical industry as identifying all protein targets is important to assess (side) effects and repositioning opportunities for a drug., Results: Here, we present a novel integrated structure- and system-based approach of drug-target prediction (iDTP) to enable the large-scale discovery of new targets for small molecules, such as pharmaceutical drugs, co-factors and metabolites (collectively called 'drugs'). For a given drug, our method uses sequence order-independent structure alignment, hierarchical clustering and probabilistic sequence similarity to construct a probabilistic pocket ensemble (PPE) that captures promiscuous structural features of different binding sites on known targets. A drug's PPE is combined with an approximation of its delivery profile to reduce false positives. In our cross-validation study, we use iDTP to predict the known targets of 11 drugs, with 63% sensitivity and 81% specificity. We then predicted novel targets for these drugs-two that are of high pharmacological interest, the peroxisome proliferator-activated receptor gamma and the oncogene B-cell lymphoma 2, were successfully validated through in vitro binding experiments. Our method is broadly applicable for the prediction of protein-small molecule interactions with several novel applications to biological research and drug development., Availability and Implementation: The program, datasets and results are freely available to academic users at http://sfb.kaust.edu.sa/Pages/Software.aspx., (© The Author 2015. Published by Oxford University Press.)

Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour.

A main source of gene expression noise in prokaryotes is translational bursting. It arises from efficient translation of mRNAs with low copy numbers, which makes the production of protein copies highly variable and pulsatile. To obtain analytical solutions, previous models to capture this noise source had to assume translation to be initiation-limited, representing the burst size by a specific type of a long-tail distribution. However, there is increasing evidence suggesting that the initiation is not the rate-limiting step in certain settings, for example, under stress conditions. Here, to overcome the limitations imposed by the initiation-limited assumption, we present a new analytical approach that can evaluate biological consequences of the protein burst size with a general distribution. Since our new model can capture the contribution of other factors to the translational noise, it can be used to analyze the effects of gene expression noise in more general settings. We used this new model to analytically analyze the connection between the burst size and the stability of gene expression processes in various settings. We found that the burst size with different distributions can lead to quantitatively and qualitatively different stability characteristics of protein abundance and can have non-intuitive effects. By allowing analysis of how the stability of gene expression processes changes based on various distributions of translational noise, our analytical approach is expected to enable deeper insights into the control of cell fate decision-making, the evolution of cryptic genetic variations, and fine-tuning of gene circuits.

Posttranslational modifications to the intracellular domain of the EGFR are known to regulate EGFR functions; however, modifications to the extracellular domain and their effects remain relatively unexplored. Here, we determined that methylation at R198 and R200 of the EGFR extracellular domain by protein arginine methyltransferase 1 (PRMT1) enhances binding to EGF and subsequent receptor dimerization and signaling activation. In a mouse orthotopic colorectal cancer xenograft model, expression of a methylation-defective EGFR reduced tumor growth. Moreover, increased EGFR methylation sustained signaling activation and cell proliferation in the presence of the therapeutic EGFR monoclonal antibody cetuximab. In colorectal cancer patients, EGFR methylation level also correlated with a higher recurrence rate after cetuximab treatment and reduced overall survival. Together, these data indicate that R198/R200 methylation of the EGFR plays an important role in regulating EGFR functionality and resistance to cetuximab treatment.

The focal adhesion kinase (FAK) and the related protein-tyrosine kinase 2-beta (Pyk2) are highly versatile multidomain scaffolds central to cell adhesion, migration, and survival. Due to their key role in cancer metastasis, understanding and inhibiting their functions are important for the development of targeted therapy. Because FAK and Pyk2 are involved in many different cellular functions, designing drugs with partial and function-specific inhibitory effects would be desirable. Here, we summarise recent progress in understanding the structural mechanism of how the tug-of-war between intramolecular and intermolecular interactions allows these protein 'nanomachines' to become activated in a site-specific manner., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.

SUMOylation has been implicated in cellular stress adaptation, but its role in regulating liver kinase B1 (LKB1), a major upstream kinase of the energy sensor AMP-activated protein kinase (AMPK), is unknown. Here, we show that energy stress triggers an increase in SUMO1 modification of LKB1, despite a global reduction in both SUMO1 and SUMO2/3 conjugates. During metabolic stress, SUMO1 modification of LKB1 lysine 178 is essential in promoting its interaction with AMPK via a SUMO-interacting motif (SIM) essential for AMPK activation. The LKB1 K178R SUMO mutant had defective AMPK signaling and mitochondrial function, inducing death in energy-deprived cells. These results provide additional insight into how LKB1-AMPK signaling is regulated during energy stress, and they highlight the critical role of SUMOylation in maintaining the cell's energy equilibrium., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The canonical action of the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI3K) is to associate with the p110α catalytic subunit to allow stimuli-dependent activation of the PI3K pathway. We elucidate a p110α-independent role of homodimerized p85α in the positive regulation of PTEN stability and activity. p110α-free p85α homodimerizes via two intermolecular interactions (SH3:proline-rich region and BH:BH) to selectively bind unphosphorylated activated PTEN. As a consequence, homodimeric but not monomeric p85α suppresses the PI3K pathway by protecting PTEN from E3 ligase WWP2-mediated proteasomal degradation. Further, the p85α homodimer enhances the lipid phosphatase activity and membrane association of PTEN. Strikingly, we identified cancer patient-derived oncogenic p85α mutations that target the homodimerization or PTEN interaction surface. Collectively, our data suggest the equilibrium of p85α monomer-dimers regulates the PI3K pathway and disrupting this equilibrium could lead to disease development.

Resistance to HER2-targeted therapies remains a major obstacle in the treatment of HER2-overexpressing breast cancer. Understanding the molecular pathways that contribute to the development of drug resistance is needed to improve the clinical utility of novel agents, and to predict the success of targeted personalized therapy based on tumor-specific mutations. Little is known about the clinical significance of HER family mutations in breast cancer. Because mutations within HER1/EGFR are predictive of response to tyrosine kinase inhibitors (TKI) in lung cancer, we investigated whether mutations in HER family kinase domains are predictive of response to targeted therapy in HER2-overexpressing breast cancer. We sequenced the HER family kinase domains from 76 HER2-overexpressing invasive carcinomas and identified 12 missense variants. Patients whose tumors carried any of these mutations did not respond to HER2 directed therapy in the metastatic setting. We developed mutant cell lines and used structural analyses to determine whether changes in protein conformation could explain the lack of response to therapy. We also functionally studied all HER2 mutants and showed that they conferred an aggressive phenotype and altered effects of the TKI lapatinib. Our data demonstrate that mutations in the finely tuned HER kinase domains play a critical function in breast cancer progression and may serve as prognostic and predictive markers., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr(925) facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr(925) phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr(861), located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser(910) by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

LD motifs (leucine-aspartic acid motifs) are short helical protein-protein interaction motifs that have emerged as key players in connecting cell adhesion with cell motility and survival. LD motifs are required for embryogenesis, wound healing and the evolution of multicellularity. LD motifs also play roles in disease, such as in cancer metastasis or viral infection. First described in the paxillin family of scaffolding proteins, LD motifs and similar acidic LXXLL interaction motifs have been discovered in several other proteins, whereas 16 proteins have been reported to contain LDBDs (LD motif-binding domains). Collectively, structural and functional analyses have revealed a surprising multivalency in LD motif interactions and a wide diversity in LDBD architectures. In the present review, we summarize the molecular basis for function, regulation and selectivity of LD motif interactions that has emerged from more than a decade of research. This overview highlights the intricate multi-level regulation and the inherently noisy and heterogeneous nature of signalling through short protein-protein interaction motifs.

Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.

The current treatment regimens for HIV include over 20 anti-retrovirals. However, adverse drug effects and the emergence of drug resistance necessitates the continued improvement of the existing drug classes as well as the development of novel drugs that target as yet therapeutically unexploited viral and cellular pathways. Here we demonstrate a strategy for the discovery of protein-protein interaction inhibitors of the viral pathogenicity factor HIV-1 Nef and its interaction with the host factor SH3. A combination of a time-resolved fluorescence resonance energy resonance energy transfer-based assay and a label-free resonant waveguide grating-based assay was optimized for high-throughput screening formats.

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc-induced through interaction with the phosphorylated receptor-releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells.

Vesicular (v)- and target (t)-SNAREs play essential roles in intracellular membrane fusion through the formation of cytoplasmic α-helical bundles. Several v-SNAREs have a Longin N-terminal extension that, by promoting a closed conformation, plays an autoinhibitory function and decreases SNARE complex formation and membrane fusion efficiency. The molecular mechanism leading to Longin v-SNARE activation is largely unknown. Here we find that exocytosis mediated by the Longin v-SNARE TI-VAMP/VAMP7 is activated by tonic treatment with insulin and insulin-like growth factor-1 but not by depolarization and intracellular calcium rise. In search of a potential downstream mechanism, we found that TI-VAMP is phosphorylated in vitro by c-Src kinase on tyrosine 45 of the Longin domain. Accordingly, a mutation of tyrosine 45 into glutamate, but not phenylalanine, activates both t-SNARE binding and exocytosis. Activation of TI-VAMP-mediated exocytosis thus relies on tyrosine phosphorylation.

The polyunsaturated fatty acid (PUFA) synthases from deep-sea bacteria invariably contain multiple acyl carrier protein (ACP) domains in tandem. This conserved tandem arrangement has been implicated in both amplification of fatty acid production (additive effect) and in structural stabilization of the multidomain protein (synergistic effect). While the more accepted model is one in which domains act independently, recent reports suggest that ACP domains may form higher oligomers. Elucidating the three-dimensional structure of tandem arrangements may therefore give important insights into the functional relevance of these structures, and hence guide bioengineering strategies. In an effort to elucidate the three-dimensional structure of tandem repeats from deep-sea anaerobic bacteria, we have expressed and purified a fragment consisting of five tandem ACP domains from the PUFA synthase from Photobacterium profundum. Analysis of the tandem ACP fragment by analytical gel filtration chromatography showed a retention time suggestive of a multimeric protein. However, small angle X-ray scattering (SAXS) revealed that the multi-ACP fragment is an elongated monomer which does not form a globular unit. Stokes radii calculated from atomic monomeric SAXS models were comparable to those measured by analytical gel filtration chromatography, showing that in the gel filtration experiment, the molecular weight was overestimated due to the elongated protein shape. Thermal denaturation monitored by circular dichroism showed that unfolding of the tandem construct was not cooperative, and that the tandem arrangement did not stabilize the protein. Taken together, these data are consistent with an elongated beads-on-a-string arrangement of the tandem ACP domains in PUFA synthases, and speak against synergistic biocatalytic effects promoted by quaternary structuring. Thus, it is possible to envision bioengineering strategies which simply involve the artificial linking of multiple ACP domains for increasing the yield of fatty acids in bacterial cultures.

Despite recent insights into melanoma genetics, systematic surveys for driver mutations are challenged by an abundance of passenger mutations caused by carcinogenic UV light exposure. We developed a permutation-based framework to address this challenge, employing mutation data from intronic sequences to control for passenger mutational load on a per gene basis. Analysis of large-scale melanoma exome data by this approach discovered six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), three of which-RAC1, PPP6C, and STK19-harbored recurrent and potentially targetable mutations. Integration with chromosomal copy number data contextualized the landscape of driver mutations, providing oncogenic insights in BRAF- and NRAS-driven melanoma as well as those without known NRAS/BRAF mutations. The landscape also clarified a mutational basis for RB and p53 pathway deregulation in this malignancy. Finally, the spectrum of driver mutations provided unequivocal genomic evidence for a direct mutagenic role of UV light in melanoma pathogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The functions of Src family kinases are tightly regulated through Src homology (SH) domain-mediated protein-protein interactions. We previously reported the biophysical characteristics of the apoptosis-linked gene 2-interacting protein X (Alix) in complex with the haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear proline-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction., (Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.)

In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate. In addition, the concepts of proximity-driven catalysis are extended to include modification of the natural Fyn SH3 domain with metallopeptides based on a known proline-rich peptide ligand. The development of orthogonal catalyst-substrate pairs for modification in lysate, and the extension of these methods to new natural protein domains, highlight the capabilities for new reaction design possible in chemical approaches to site-specific protein modification.

Noise caused by stochastic fluctuations in genetic circuits (transcription and translation) is now appreciated as a central aspect of cell function and phenotypic behavior. Noise has also been detected in signaling networks, but the origin of this noise and how it shapes cellular outcomes remain poorly understood. Here, we argue that noise in signaling networks results from the intrinsic promiscuity of protein-protein interactions (PPIs), and that this noise has shaped cellular signal transduction. Features promoted by the presence of this molecular signaling noise include multimerization and clustering of signaling components, pleiotropic effects of gross changes in protein concentration, and a probabilistic rather than a linear view of signal propagation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Downregulation of major histocompatibility complex class I (MHC-I) by HIV-1 Nef protein is indispensable for evasion of protective immunity by HIV-1. Though it has been suggested that the N-terminal region of Nef contributes to the function by associating with a mu-1A subunit of adaptor protein 1, the structural basis of the interaction between Nef and mu-1A remains elusive. We found that a tripartite hydrophobic motif (Trp13/Val16/Met20) in the N terminus of Nef was required for the MHC-I downregulation. Importantly, the motif functioned as a noncanonical mu-1A-binding motif for the interaction with the tyrosine motif-binding site of the mu-1A subunit. Our findings will help understanding of how HIV-1 evades the antiviral immune response by selectively redirecting the cellular protein trafficking system.

The HIV-1 auxiliary protein Nef is required for the onset and progression of AIDS in HIV-1-infected persons. Here, we have deciphered the mode of action of a second-generation inhibitor of Nef, DLC27-14, presenting a competitive IC(50) of ∼16 μM measured by MALDI-TOF experiments. Thermal protein denaturation experiments revealed a negative effect on stability of Nef in the presence of a saturating concentration of the inhibitor. The destabilizing action of DLC27-14 was confirmed by a HIV protease-based experiment, in which the protease sensitivity of DLC27-14-bound Nef was three times as high as that of apo Nef. The only compatible docking modes of action for DLC27-14 suggest that DLC27-14 promotes an opening of two α-helices that would destabilize the Nef core domain. DLC27-14 thus acts as a specific protein disorder catalyzer that destabilizes the folded conformation of the protein. Our results open novel avenues toward the development of next-generation Nef inhibitors., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Focal adhesion kinase (FAK) has an astonishing number of ligands and functions, which enable it to contribute to embryonic development and human health. FAK can promote different effects in similar cellular environments or similar effects in different cellular environments. Recent advances in structural and cellular analysis of FAK are starting to reveal the interrelationships between the conformations, localizations, interactions, and functions of FAK. This review focuses on our emerging understanding of how the structural framework of FAK mechanistically allows it to integrate manifold stimuli into environment-specific functions., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In Escherichia coli, cytokinesis is orchestrated by FtsZ, which forms a Z-ring to drive septation. Spatial and temporal control of Z-ring formation is achieved by the Min and nucleoid occlusion (NO) systems. Unlike the well-studied Min system, less is known about the anti-DNA guillotining NO process. Here, we describe studies addressing the molecular mechanism of SlmA (synthetic lethal with a defective Min system)-mediated NO. SlmA contains a TetR-like DNA-binding fold, and chromatin immunoprecipitation analyses show that SlmA-binding sites are dispersed on the chromosome except the Ter region, which segregates immediately before septation. SlmA binds DNA and FtsZ simultaneously, and the SlmA-FtsZ structure reveals that two FtsZ molecules sandwich a SlmA dimer. In this complex, FtsZ can still bind GTP and form protofilaments, but the separated protofilaments are forced into an anti-parallel arrangement. This suggests that SlmA may alter FtsZ polymer assembly. Indeed, electron microscopy data, showing that SlmA-DNA disrupts the formation of normal FtsZ polymers and induces distinct spiral structures, supports this. Thus, the combined data reveal how SlmA derails Z-ring formation at the correct place and time to effect NO.