Crystal structures of the Gon7/Pcc1 and Bud32/Cgi121 complexes provide a model for the complete yeast KEOPS complex.

International audienceThe yeast KEOPS protein complex comprising Kae1, Bud32, Cgi121, Pcc1 and Gon7 is responsible for the essential tRNA threonylcarbamoyladenosine (t(6)A) modification. Deletion of genes coding for the KEOPS subunits also affects telomere elongation and transcriptional regulation. In the present work, the crystal structure of Bud32/Cgi121 in complex with ADP revealed that ADP is bound in the catalytic site of Bud32 in a canonical manner characteristic of Protein Kinase A (PKA) family proteins. We found that Gon7 forms a stable heterodimer with Pcc1 and report the crystal structure of the Pcc1-Gon7 heterodimer. Gon7 interacts with the same Pcc1 region engaged in the archaeal Pcc1 homodimer. We further show that yeast KEOPS, unlike its archaeal counterpart, exists as a heteropentamer in which Gon7, Pcc1, Kae1, Bud32 and Cgi121 also adopt a linear arrangement. We constructed a model of yeast KEOPS that provides structural insight into the role of Gon7. The model also revealed the presence of a highly positively charged crater surrounding the entrance of Kae1 that likely binds tRNA

The Structure of the SlrP-hTrx1 Complex Sheds Light on the Autoinhibition Mechanism of the Type-III Secretion System Effectors of the NEL Family

Salmonella infections are a leading cause of bacterial foodborne illness in the United States and the European Union. Antimicrobial therapy is often administered to treat the infection but increasing isolates are being detected that demonstrate resistance to multiple antibiotics. Salmonella enterica contains two virulence related type-III secretion systems (T3SS): one promotes invasion of the intestine and the other one mediates systemic disease. Both of them secrete the SlrP protein acting as E3 ubiquitin ligase in human host cells where it targets thioredoxin-1 (Trx1). SlrP belongs to the NEL family of bacterial E3 ubiquitin ligases that have been observed in two distinct autoinhibitory conformations. We solved the 3D structure of the SlrP/Trx1 complex and determined the Trx1 ubiquitination site. The description of the substrate-binding mode sheds light on the first step of the activation mechanism of SlrP. Comparison with the available structural data of other NEL effectors allowed us to gain new insights into their autoinhibitory mechanism. We propose a molecular mechanism for the regulation of SlrP in which structural constraints sequestrating the NEL domain would be sequentially released. This work thus constitutes a new milestone in the understanding of how these T3SS effectors influence pathogen virulence. It also provides the fundamental basis for future development of new antimicrobials.Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, Spain. Grant P08-CVI-03487Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund. SAF2010-15015 and SAF2013-46229-RSpanish Ministry of Science and Innovation. Grant FR2009-0103Programme Picasso-2010 from the Partenariat Hubert Curie

The crystal structure of Trz1, the long form RNase Z from yeast.

tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3΄ maturation of tRNAs in all domains of life. It is a member of the β-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused β-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two β-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z

CCN3: a key growth regulator in Chronic Myeloid Leukaemia

Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active Bcr-Abl tyrosine kinase. We have shown previously that the negative growth regulator, CCN3, is down-regulated as a result of Bcr-Abl kinase activity and that CCN3 has a reciprocal relationship of expression with BCR-ABL. We now show that CCN3 confers growth regulation in CML cells by causing growth inhibition and regaining sensitivity to the induction of apoptosis. The mode of CCN3 induced growth regulation was investigated in K562 CML cells using gene transfection and treatment with recombinant CCN3. Both strategies showed CCN3 regulated CML cell growth by reducing colony formation capacity, increasing apoptosis and reducing ERK phosphorylation. K562 cells stably transfected to express CCN3 showed enhanced apoptosis in response to treatment with the tyrosine kinase inhibitor, imatinib. Whilst CCN3 expression was low or undetectable in CML stem cells, primary CD34+ CML progenitors were responsive to treatment with recombinant CCN3. This study shows that CCN3 is an important growth regulator in haematopoiesis, abrogation of CCN3 expression enhances BCR-ABL dependent leukaemogenesis. CCN3 restores growth regulation, regains sensitivity to the induction of apoptosis and enhances imatinib cell kill in CML cells. CCN3 may provide an additional therapeutic strategy in the management of CML

Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases

Nov/Ccn3, a novel transcriptional target of FoxO1, impairs pancreatic β-cell function.

Type 2 diabetes is characterized by both insulin resistance and progressive deterioration of β-cell function. The forkhead transcription factor FoxO1 is a prominent mediator of insulin signaling in β-cells. We reasoned that identification of FoxO1 target genes in β-cells could reveal mechanisms linking β-cell dysfunction to insulin resistance. In this study, we report the characterization of Nov/Ccn3 as a novel transcriptional target of FoxO1 in pancreatic β-cells. FoxO1 binds to an evolutionarily conserved response element in the Ccn3 promoter to regulate its expression. Accordingly, CCN3 levels are elevated in pancreatic islets of mice with overexpression of a constitutively active form of FoxO1 or insulin resistance. Our functional studies reveal that CCN3 impairs β-cell proliferation concomitantly with a reduction in cAMP levels. Moreover, CCN3 decreases glucose oxidation, which translates into inhibition of glucose-stimulated Ca(2+) entry and insulin secretion. Our results identify CCN3, a novel transcriptional target of FoxO1 in pancreatic β-cells, as a potential target for therapeutic intervention in the treatment of diabetes

Minor Impact of A258D Mutation on Biochemical and Enzymatic Properties of Leishmania infantum GDP-Mannose Pyrophosphorylase

Background: Leishmaniasis, a vector-borne disease caused by the protozoan parasite from the genus Leishmania, is endemic to tropical and subtropical areas. Few treatments are available against leishmaniasis, with all presenting issues of toxicity, resistance, and/or cost. In this context, the development of new antileishmanial drugs is urgently needed. GDP-mannose pyrophosphorylase (GDP-MP), an enzyme involved in the mannosylation pathway, has been described to constitute an attractive therapeutic target for the development of specific antileishmanial agents. Methods: In this work, we produced, purified, and analyzed the enzymatic properties of the recombinant L. infantum GDP-MP (LiGDP-MP), a single leishmanial GDP-MP that presents mutation of an aspartate instead of an alanine at position 258, which is also the single residue difference with the homolog in L. donovani: LdGDP-MP. Results: The purified LiGDP-MP displayed high substrate and cofactor specificities, a sequential random mechanism of reaction, and the following kinetic constants: Vm at 0.6 µM·min−1, Km from 15–18 µM, kcat from 12.5–13 min−1, and kcat/Km at around 0.8 min−1µM−1. Conclusions: These results show that LiGDP-MP has similar biochemical and enzymatic properties to LdGDP-MP. Further studies are needed to determine the advantage for L. infantum of the A258D residue change in GDP-MP

CCN3 suppresses mitogenic signalling and reinstates growth control mechanisms in Chronic Myeloid Leukaemia

CCN3, a tumour suppressor gene, is down-regulated as a result of BCR-ABL tyrosine kinase activity in Chronic Myeloid Leukaemia (CML). We have established a stable CCN3 expression model in the human K562 CML cell line and have further validated the role for CCN3 in the leukaemogenic process. K562 cells stably transfected with CCN3 (K562/CCN3; 2.25 × 106 copies per 50 ng cDNA) demonstrated over 50% reduction in cell growth in comparison to cells stably transfected with empty vector (K562/control; p = 0.005). K562/CCN3 cells had reduced colony formation capacity (reduced by 29.7%, p = 0.03) and reduced mitogenic signalling in comparison to K562/control cells (reduced by 29.5% (p = 0.002) and 37.4% (p = 0.017) for phosphorylation levels of ERK and AKT respectively). K562/CCN3 cells showed an accumulation of events within the subG0 phase of the cell cycle and increased apoptosis was confirmed by a three-fold increase in annexin V binding (p < 0.05). K562/CCN3 cells exposed to Imatinib (1 μM and 5 μM) showed an increase in events within the subG0 phase of cell cycle over 96 h and mirrored the enhanced cell kill demonstrated by Annexin staining. Wild type K562 cells treated with recombinant human Ccn3 (10 nM) in combination with Imatinib (5 μM) also displayed enhanced cell kill (p = 0.008). K562/CCN3 cells displayed increased adhesion to matrigel™ (2.92 ± 0.52 fold increase compared to K562/control) which was commensurate with increased expression of the alpha 6 and beta 4 integrins (6.53 ± 0.47 and 1.94 ± 0.07 fold increase in gene expression respectively (n = 3, p < 0.05)). CCN3 restores cellular growth regulatory properties that are absent in CML and sensitises CML cells to imatinib induced apoptosis. CCN3 may provide novel avenues for the development of alternate therapeutic strategies