scholarly journals Structural insights into the sequence-specific recognition of Piwi by Drosophila Papi

2018 ◽  
Vol 115 (13) ◽  
pp. 3374-3379 ◽  
Author(s):  
Yuhan Zhang ◽  
Weiwei Liu ◽  
Ronghong Li ◽  
Jiaqi Gu ◽  
Ping Wu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Biochemical Analysis ◽  
Critical Role ◽  
Direct Interaction ◽  
Structural Basis ◽  
Binding Partner ◽  
Transposon Silencing ◽  
Tudor Domain ◽  
Structural Insights

The Tudor domain-containing (Tdrd) family proteins play a critical role in transposon silencing in animal gonads by recognizing the symmetrically dimethylated arginine (sDMA) on the (G/A)R motif of the N-terminal of PIWI family proteins via the eTud domains. Papi, also known as “Tdrd2,” is involved in Zucchini-mediated PIWI-interacting RNA (piRNA) 3′-end maturation. Intriguingly, a recent study showed that, in papi mutant flies, only Piwi-bound piRNAs increased in length, and not Ago3-bound or Aub-bound piRNAs. However, the molecular and structural basis of the Papi–Piwi complex is still not fully understood, which limits mechanistic understanding of the function of Papi in piRNA biogenesis. In the present study, we determined the crystal structures of Papi-eTud in the apo form and in complex with a peptide containing unmethylated or dimethylated R10 residues. Structural and biochemical analysis showed that the Papi interaction region on the Drosophila Piwi contains an RGRRR motif (R7–R11) distinct from the consensus (G/A)R motif recognized by canonical eTud. Mass spectrometry results indicated that Piwi is the major binding partner of Papi in vivo. The papi mutant flies suffered from both fertility and transposon-silencing defects, supporting the important role conferred to Papi in piRNA 3′ processing through direct interaction with Piwi proteins.

scholarly journals Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

2006 ◽  
Vol 398 (3) ◽  
pp. 353-360 ◽  
Author(s):  
Jingzhi Li ◽  
Yunkun Wu ◽  
Xinguo Qian ◽  
Bingdong Sha
Keyword(s):  
Crystal Structure ◽  
Close Contact ◽  
Critical Role ◽  
Peptide Binding ◽  
Structural Basis ◽  
Cellular Processes ◽  
Terminal Form ◽  
Charged Residues ◽  
Domain I

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40–Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a β-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge–charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.

Wnt5a Induces Association of ROR1 with 14-3-3ζ to Enhance Chemotaxis and Proliferation in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 349-349 ◽  
Author(s):  
Jian Yu ◽  
Liguang Chen ◽  
Yun Chen ◽  
Ling Zhang ◽  
Laura Z. Rassenti ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Research Funding ◽  
Critical Role ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Binding Motif ◽  
Human Leukemia

Abstract Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic antigen that is expressed on CLL cells, but not on normal postpartum tissues. We found that ROR1 was a receptor for Wnt5a, which could activate Rho GTPases (e.g. RhoA and Rac1) in CLL cells by inducing the recruitment to ROR1 of guanine exchange factors (GEFs), notably ARHGEF2. How ARHGEF2 can complex with ROR1 was not known. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 and identified 14-3-3ζ, a highly conserved, cytoplasmic-protein member of the tetratricopeptide repeat-like superfamily. 14-3-3ζ plays a critical role in cell-signaling pathways, which promote proliferation, adhesion, and survival in a variety of human cancers. We validated the Wnt5a-induced interaction of ROR1 with 14-3-3ζ in primary CLL cells using co-immunoprecipitation studies and immunoblot analyses. We found the capacity of Wnt5a to induce ROR1 to associate with 14-3-3ζ could be blocked by cirmtuzumab, a first-in-class humanized mAb specific for a functional epitope in the ROR1-extracellular domain; this mAb is undergoing clinical testing in patients with CLL. Furthermore, we found that 14-3-3ζ could interact with ARHGEF2 in CLL cells. Silencing 14-3-3ζ via RNAi impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance the in vitro exchange activity of ARHGEF2 for RhoA and Rac1, and (3) induce activation of RhoA and Rac1 in primary CLL cells. Consistent with these findings, we found that Difopein, an inhibitor of 14-3-3ζ, also could inhibit Wnt5a-enhanced chemokine-directed migration and proliferation of primary CLL cells in vitro, at low concentrations that did not result in leukemia-cell apoptosis. To examine structure-function relationships, we employed the MEC1 cell line, which was derived from human CLL. Prior studies found MEC1 cells expressed Wnt5a, which induced activation of RhoA and Rac1 in MEC1 cells made to express ROR1 (MEC1-ROR1), but not in parental MEC1 cells, which lacked ROR1. Similar to work on primary CLL cells, we detected 14-3-3ζ in anti-ROR1 immune precipitates via mass spectrometry and immunoblot analyses. We identified a 14-3-3ζ binding motif (RSPS857SAS) in the cytoplasmic domain of ROR1; site directed mutagenesis and transfection of MEC1 with mutant forms of ROR1 determined that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2-dependent activation of RhoA and Rac1, respectively. In addition, we used CRISPR/Cas9 technology to delete 14-3-3ζ (Δ14-3-3ζ) in MEC1 and MEC1-ROR1 cells. We found that MEC1-ROR1 had significantly higher rates of proliferation than MEC1-ROR1-Δ14-3-3ζ cells, which in turn had rates of proliferation comparable to those of MEC1 cells or MEC1-Δ14-3-3ζ lacking expression of ROR1. MEC1-ROR1 also had a significantly greater capacity to migrate in response to chemokine (CCL21) than did MEC1-ROR1-Δ14-3-3ζ cells, which migrated in response to CCL21 as well as MEC1-Δ14-3-3ζ or MEC1 cells lacking expression of ROR1. To examine whether such differences affected leukemia-cell growth in vivo, we engrafted Rag2−/−γc−/− mice each with equal numbers of MEC1, MEC1-Δ14-3-3ζ, MEC1-ROR1, or MEC1-ROR1-Δ14-3-3ζ cells. We found that mice that received MEC1-ROR1 cells had significantly higher levels of engraftment and human leukemia-cell proliferation than did mice that received MEC1, MEC1-Δ14-3-3ζ, or ROR1-Δ14-3-3ζ cells, which had comparable levels of engraftment and proliferation, indicating that 14-3-3ζ was necessary for the engraftment/growth advantage of MEC1-ROR1 over MEC1 cells in vivo. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1-dependent-signaling leading to enhanced migration and proliferation of CLL cells in vitro and in vivo. Disclosures Kipps: Roche: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding.

scholarly journals The Structural Basis for Glycerol Permeation by human AQP7

2019 ◽  
Author(s):  
Li Zhang ◽  
Deqiang Yao ◽  
Fu Zhou ◽  
Qing Zhang ◽  
Ying Xia ◽  
...  
Keyword(s):  
Physiological Condition ◽  
Critical Role ◽  
Binding Pocket ◽  
Structural Basis ◽  
Glycerol Metabolism ◽  
Functional Assay ◽  
Substrate Binding Pocket ◽  
Glycerol Molecule ◽  
Cytoplasmic Side

AbstractHuman glycerol channel AQP7 conducts glycerol release from adipocyte and entry into the cells in pancreatic islets, muscles and kidney tubule, and thus regulate glycerol metabolism in those tissues. Compared with other human aquaglyceroporins, AQP7 shows a less conserved “NPA” motif in the center cavity, and a pair of aromatic residues at Ar/R selectivity filter. To understand the structural basis for the glycerol conductance, we crystallized the human AQP7 and determined the structure at 3.7 Å. A substrate binding pocket was found near to the Ar/R filter and the bound glycerol molecule stabilized by R229. In vivo functional assay on human AQP7 as well as AQP3 and AQP10 demonstrated strong glycerol transportation activities at physiological condition. The human AQP7 structure reveals a fully closed conformation with its permeation pathway strictly confined by Ar/R filter at the exoplasmic side and the gate at the cytoplasmic side, and the dislocation of the residues at narrowest parts of glycerol pathway in AQP7 play a critical role in controlling the glycerol flux.

scholarly journals Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex.

Genetics ◽  
1993 ◽  
Vol 133 (2) ◽  
pp. 183-191 ◽  
Author(s):  
M P Longhese ◽  
L Jovine ◽  
P Plevani ◽  
G Lucchini
Keyword(s):  
Dna Polymerase ◽  
Large Subunit ◽  
Spontaneous Mutation ◽  
Critical Role ◽  
Direct Interaction ◽  
Small Subunit ◽  
Dna Polymerase Alpha ◽  
Dna Primase ◽  
Primase Complex

Abstract Different pri1 and pri2 conditional mutants of Saccharomyces cerevisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show an enhanced rate of both mitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity of their defects in cell growth and DNA synthesis. These effects might be attributable to the formation of nicked and gapped DNA molecules that are substrates for recombination and error-prone repair, due to defective DNA replication in the primase mutants. Furthermore, pri1 and pri2 mutations inhibit sporulation and affect spore viability, with the unsporulated mutant cells arresting with a single nucleus, suggesting that DNA primase plays a critical role during meiosis. The observation that all possible pairwise combinations of two pri1 and two pri2 alleles are lethal provides further evidence for direct interaction of the primase subunits in vivo. Immunopurification and immunoprecipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase alpha, and either mediates or stabilizes association of the p48 polypeptide in the DNA polymerase alpha-primase complex.

scholarly journals Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates

2019 ◽  
Vol 116 (8) ◽  
pp. 2919-2924 ◽  
Author(s):  
Xiao Zhang ◽  
Lian-Huan Wei ◽  
Yuxin Wang ◽  
Yu Xiao ◽  
Jun Liu ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Catalytic Mechanism ◽  
Tertiary Structure ◽  
Structural Basis ◽  
Multiple Substrates ◽  
Functional Studies ◽  
Structural Insights ◽  
Demethylation Activity

FTO demethylates internal N6-methyladenosine (m6A) and N6,2′-O-dimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate—in vitro and in vivo—that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am. Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A and m6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

scholarly journals Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

2010 ◽  
Vol 84 (16) ◽  
pp. 8072-8084 ◽  
Author(s):  
Sarah E. Hobdey ◽  
Brian J. Kempf ◽  
Benjamin P. Steil ◽  
David J. Barton ◽  
Olve B. Peersen
Keyword(s):  
Amino Acid ◽  
Rna Binding ◽  
Critical Role ◽  
Polymerase Activity ◽  
Structural Basis ◽  
Wild Type ◽  
Elongation Complex ◽  
Virus Growth ◽  
The Stability

ABSTRACT The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.

The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) regulates the production of proangiogenic molecules by myeloma cells and suppresses hypoxia-inducible factor-1 α (HIF-1α) activity: involvement in myeloma-induced angiogenesis

Blood ◽  
2007 ◽  
Vol 110 (13) ◽  
pp. 4464-4475 ◽  
Author(s):  
Simona Colla ◽  
Sara Tagliaferri ◽  
Francesca Morandi ◽  
Paolo Lunghi ◽  
Gaetano Donofrio ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Family Member ◽  
Tumor Suppressor Gene ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Hypoxia Inducible Factor ◽  
Direct Interaction ◽  
Suppressor Gene

Angiogenesis has a critical role in the pathophysiology of multiple myeloma (MM); however, the molecular mechanisms underlying this process are not completely elucidated. The new tumor-suppressor gene inhibitor of growth family member 4 (ING4) has been recently implicated in solid tumors as a repressor of angiogenesis. In this study, we found that ING4 expression in MM cells was correlated with the expression of the proangiogenic molecules interleukin-8 (IL-8) and osteopontin (OPN). Moreover, we demonstrate that ING4 suppression in MM cells up-regulated IL-8 and OPN, increasing the hypoxia inducible factor-1α (HIF-1α) activity and its target gene NIP-3 expression in hypoxic condition. In turn, we show that the inhibition of HIF-1α by siRNA suppressed IL-8 and OPN production by MM cells under hypoxia. A direct interaction between ING4 and the HIF prolyl hydroxylase 2 (HPH-2) was also demonstrated. Finally, we show that ING4 suppression in MM cells significantly increased vessel formation in vitro, blunted by blocking IL-8 or OPN. These in vitro observations were confirmed in vivo by finding that MM patients with high IL-8 production and microvascular density (MVD) have significantly lower ING4 levels compared with those with low IL-8 and MVD. Our data indicate that ING4 exerts an inhibitory effect on the production of proangiogenic molecules and consequently on MM-induced angiogenesis.

scholarly journals Gemin4

2000 ◽  
Vol 148 (6) ◽  
pp. 1177-1186 ◽  
Author(s):  
Bernard Charroux ◽  
Livio Pellizzoni ◽  
Robert A. Perkinson ◽  
Jeongsik Yong ◽  
Andrej Shevchenko ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Muscular Atrophy ◽  
Critical Role ◽  
Direct Interaction ◽  
Nuclear Bodies ◽  
Dead Box ◽  
Core Proteins ◽  
Smn Complex ◽  
Smn Protein

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.

scholarly journals Protein engineering expands the effector recognition profile of a rice NLR immune receptor

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Juan Carlos De la Concepcion ◽  
Marina Franceschetti ◽  
Dan MacLean ◽  
Ryohei Terauchi ◽  
Sophien Kamoun ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Direct Interaction ◽  
Structural Basis ◽  
In Planta ◽  
Pathogen Effectors ◽  
Binding Interface ◽  
Pathogen Effector ◽  
Integrated Domains

Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.

scholarly journals Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2

2017 ◽  
Vol 114 (47) ◽  
pp. 12483-12488 ◽  
Author(s):  
Heng Zhang ◽  
Ke Liu ◽  
Natsuko Izumi ◽  
Haiming Huang ◽  
Deqiang Ding ◽  
...  
Keyword(s):  
Binding Mode ◽  
Arginine Methylation ◽  
P Element ◽  
Structural Basis ◽  
Dependent Manner ◽  
Transposon Silencing ◽  
Tudor Domain ◽  
Genome Protection ◽  
Pirna Pathway

The P-element–induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway plays a central role in transposon silencing and genome protection in the animal germline. A family of Tudor domain proteins regulates the piRNA pathway through direct Tudor domain–PIWI interactions. Tudor domains are known to fulfill this function by binding to methylated PIWI proteins in an arginine methylation-dependent manner. Here, we report a mechanism of methylation-independent Tudor domain–PIWI interaction. Unlike most other Tudor domains, the extended Tudor domain of mammalian Tudor domain-containing protein 2 (TDRD2) preferentially recognizes an unmethylated arginine-rich sequence from PIWI-like protein 1 (PIWIL1). Structural studies reveal an unexpected Tudor domain-binding mode for the PIWIL1 sequence in which the interface of Tudor and staphylococcal nuclease domains is primarily responsible for PIWIL1 peptide recognition. Mutations disrupting the TDRD2–PIWIL1 interaction compromise piRNA maturation via 3′-end trimming in vitro. Our work presented here reveals the molecular divergence of the interactions between different Tudor domain proteins and PIWI proteins.

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