Rustless translation

ATP binding cassette proteins are a large and diverse family of molecular machines and include transmembrane transporter, chromosome maintenance and DNA repair proteins, and translation factors. However, the function of the ABCE1, the only member of subfamily E of ABC proteins, remained mysterious for over a decade, even though it is perhaps the most conserved ABC protein in eukaryotes and archaea. Recent results have now identified ABCE1 as the ribosome-recycling factor of eukaryotes and archaea. Thus, two iron-sulfur clusters - the hallmark feature of ABCE1 - help catalyze an integral step of the translational cycle at the core of the protein synthesis machinery

Vascular endothelial growth factor production and regulation in rodent and human pituitary tumor cells in vitro

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope alpha T3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-cc (TGF-a), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and aT3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-alpha, PACAP-38, and 17 beta -estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas

Crystal Structure of Human TWEAK in Complex with the Fab Fragment of a Neutralizing Antibody Reveals Insights into Receptor Binding.

The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases

Distinct requirements for the Rad32(Mre¹¹) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA

For a cancer cell to resist treatment with drugs that trap topoisomerases covalently on the DNA, the topoisomerase must be removed. In this study, we provide evidence that the Schizosaccharomyces pombe Rad32Mre11 nuclease activity is involved in the removal of both Top2 from 5′ DNA ends as well as Top1 from 3′ ends in vivo. A ctp1CtIP deletion is defective for Top2 removal but overproficient for Top1 removal, suggesting that Ctp1CtIP plays distinct roles in removing topoisomerases from 5′ and 3′ DNA ends. Analysis of separation of function mutants suggests that MRN-dependent topoisomerase removal contributes significantly to resistance against topoisomerase-trapping drugs. This study has important implications for our understanding of the role of the MRN complex and CtIP in resistance of cells to a clinically important group of anticancer drugs

Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway

Molecular pathogenesis and targeted therapy of sporadic pancreatic neuroendocrine tumors

Over the past few years, knowledge regarding the molecular pathology of sporadic pancreatic neuroendocrine tumors (PNETs) has increased substantially, and a number of targeted agents have been tested in clinical trials in this tumor type. For some of these agents there is a strong biological rationale. Among them, the mammalian target of rapamycin inhibitor Everolimus and the antiangiogenic agent Sunitinib have both been approved for the treatment of PNETs. However, there is lack of knowledge regarding biomarkers able to predict their efficacy, and mechanisms of resistance. Other angiogenesis inhibitors, such as Pazopanib, inhibitors of Src, Hedgehog or of PI3K might all be useful in association or sequence with approved agents. On the other hand, the clinical significance, and potential for treatment of the most common mutations occurring in sporadic PNETs, in the MEN-1 gene and in ATRX and DAXX, remains uncertain. The present paper reviews the main molecular changes occurring in PNETs and how they might be linked with treatment options

Snf2 family ATPases and DExx box helicases:differences and unifying concepts from high-resolution crystal structures

Proteins with sequence similarity to the yeast Snf2 protein form a large family of ATPases that act to alter the structure of a diverse range of DNA–protein structures including chromatin. Snf2 family enzymes are related in sequence to DExx box helicases, yet they do not possess helicase activity. Recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves ATP-dependent translocation on DNA. Crystal structures suggest that these enzymes travel along the minor groove, a process that can generate the torque or energy in remodelling processes. We review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both Snf2 family and DExx box helicases

Architectures and mechanisms of ATP binding cassette proteins

ATP binding cassette (ABC) ATPases form chemo-mechanical engines and switches that function in a broad range of biological processes. Most prominently, a very large family of integral membrane NTPases-ABC transporters-catalyzes the import or export of a diverse molecules across membranes. ABC proteins are also important components of the chromosome segregation, recombination, and DNA repair machineries and regulate or catalyze critical steps of ribosomal protein synthesis. Recent structural and mechanistic studies draw interesting architectural and mechanistic parallels between diverse ABC proteins. Here, I review this state of our understanding how NTP-dependent conformational changes of ABC proteins drive diverse biological processes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 492-504, 2016

ATP puts the brake on DNA double-strand break repair

DNA double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage and can result in cell inviability or chromosomal aberrations. The Mre11-Rad50-Nbs1 (MRN) ATPase-nuclease complex is a central player in the cellular response to DSBs and is implicated in the sensing and nucleolytic processing of DSBs, as well as in DSB signaling by activating the cell cycle checkpoint kinase ATM. ATP binding to Rad50 switches MRN from an open state with exposed Mre11 nuclease sites to a closed state with partially buried nuclease sites. The functional meaning of this switch remained unclear. A new study shows that ATP binding to Rad50 promotes DSB recognition, tethering, and ATM activation, while ATP hydrolysis opens the nuclease active sites to promote processing of DSBs. MRN thus emerges as functional switch that may coordinate the temporal transition from signaling to processing of DSBs