14 research outputs found

    Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein

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    Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand-protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MPB) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues

    Comparative Genomics of Peroxisome Biogenesis Proteins:Making Sense of the PEX Proteins

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    PEX genes encode proteins involved in peroxisome biogenesis and proliferation. Using a comparative genomics approach, we clarify the evolutionary relationships between the 37 known PEX proteins in a representative set of eukaryotes, including all common model organisms, pathogenic unicellular eukaryotes and human. A large number of previously unknown PEX orthologs were identified. We analyzed all PEX proteins, their conservation and domain architecture and defined the core set of PEX proteins that is required to make a peroxisome. The molecular processes in peroxisome biogenesis in different organisms were put into context, showing that peroxisomes are not static organelles in eukaryotic evolution. Organisms that lack peroxisomes still contain a few PEX proteins, which probably play a role in alternative processes. Finally, the relationships between PEX proteins of two large families, the Pex11 and Pex23 families, were analyzed, thereby contributing to the understanding of their complicated and sometimes incorrect nomenclature. We provide an exhaustive overview of this important eukaryotic organelle

    Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA

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    (Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate-binding cassette transporter OpuA restores the cell volume by accumulating large amounts of compatible solute. OpuA is gated by ionic strength and inhibited by the second messenger cyclic-di-AMP, a molecule recently shown to affect many cellular processes. Despite the master regulatory role of cyclic-di-AMP, structural and functional insights into how the second messenger regulates (transport) proteins on the molecular level are lacking. Here, we present high-resolution cryo-electron microscopy structures of OpuA and in vitro activity assays that show how the osmoregulator OpuA is activated by high ionic strength and how cyclic-di-AMP acts as a backstop to prevent unbridled uptake of compatible solutes

    Stereoselective Protection-Free Modification of 3-Keto-saccharides

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    Unprotected 3-keto-saccharides have become readily accessible via site-selective oxidation, but their protection-free functionalization is relatively unexplored. Here we show that protecting groups are obsolete in a variety of stereoselective modifications of our model substrate methyl α-glucopyranoside. This allows the preparation of rare sugars and the installation of click handles and reactive groups. To showcase the applicability of the methodology, maltoheptaose has been converted into a chemical probe, and the rare sugar evalose has been synthesized

    Is there a decline in verbal working memory over age?

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    In this study, the new standard computerized version of the reading span test was used to investigate the development of verbal working memory over age. A significant higher reading span and faster reaction times were expected for the young adults compared to the old adults, based on the processing resource theory (Just & Carpenter, 1992) and the theory of cognitive aging (Salthouse, 1994, 1996), and this hypothesis was confirmed. The new methodology made it possible to test whether there was an age-related increase in intrusion errors, which could be expected based on the inhibition theory of cognitive aging (Hasher & Zacks, 1988). The results showed that older adults made more intrusion errors than young adults thereby confirming the inhibition theory. Finally, the analysis of the memory-pattern showed a clear recency-effect for the young-, but not for the old adults. Interestingly, this has never been reported in literature before. Although more research is needed before any firm conclusions can be drawn, this decline in recency-effect shows that there are larger age-related effects in short term memory span than was expected on the basis of aging theories so far

    Novel targeting assay uncovers targeting information within peroxisomal ABC transporter Pxa1

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    The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1–95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1–95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal

    CCDC 1995084: Experimental Crystal Structure Determination

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    Related Article: Nittert Marinus, Nabil Tahiri, Margherita Duca, L. M. C. Marc Mouthaan, Simona Bianca, Marco van den Noort, Bert Poolman, Martin D. Witte, Adriaan J. Minnaard|2020|Org.Lett.|22|5622|doi:10.1021/acs.orglett.0c0198

    CCDC 1995088: Experimental Crystal Structure Determination

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    Related Article: Nittert Marinus, Nabil Tahiri, Margherita Duca, L. M. C. Marc Mouthaan, Simona Bianca, Marco van den Noort, Bert Poolman, Martin D. Witte, Adriaan J. Minnaard|2020|Org.Lett.|22|5622|doi:10.1021/acs.orglett.0c0198

    Alternating Treatment With Pazopanib and Everolimus vs Continuous Pazopanib to Delay Disease Progression in Patients With Metastatic Clear Cell Renal Cell Cancer The ROPETAR Randomized Clinical Trial

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    IMPORTANCE To our knowledge, this is the first randomized clinical trial evaluating an alternating treatment regimen in an attempt to delay disease progression in clear cell renal cell carcinoma. OBJECTIVE To test our hypothesis that an 8-week rotating treatment schedule with pazopanib and everolimus delays disease progression, exhibits more favorable toxic effects, and improves quality of life when compared with continuous treatment with pazopanib. DESIGN, SETTING, AND PARTICIPANTS This was an open-label, randomized (1: 1) study (ROPETAR trial). In total, 101 patients with treatment-naive progressivemetastatic clear cell renal cell carcinoma were enrolled between September 2012 and April 2014 from 17 large peripheral or academic hospitals in The Netherlands and followed for at least one year. INTERVENTIONS First-line treatment consisted of either an 8-week alternating treatment schedule of pazopanib 800 mg/d and everolimus 10 mg/d (rotating arm) or continuous pazopanib 800 mg/d (control arm) until progression. After progression, patients made a final rotation to either pazopanib or everolimus monotherapy (rotating arm) or initiated everolimus (control arm). MAIN OUTCOME AND MEASURES The primary end pointwas survival until first progression or death. Secondary end points included time to second progression or death, toxic effects, and quality of life. RESULTS A total of 52 patients were randomized to the rotating arm (median [range] age, 65 [44-87] years) and 49 patients to the control arm (median [range] age, 67 [38-82] years). Memorial Sloan Kettering Cancer Center risk category was favorable in 26% of patients, intermediate in 58%, and poor in 15%. Baseline characteristics and risk categories were well balanced between arms. One-year PFS1 for rotating treatment was 45%(95% CI, 33-60) and 32%(95% CI, 21-49) for pazopanib (control). Median time until first progression or death for rotating treatment was 7.4 months (95% CI, 5.6-18.4) and 9.4 months (95% CI, 6.6-11.9) for pazopanib (control) (P = .37). Mucositis, anorexia, and dizziness were more prevalent in the rotating arm during first-line treatment. No difference in quality of life was observed. CONCLUSIONS AND RELEVANCE Rotating treatment did not result in prolonged progression-free-survival, fewer toxic effects, or improved quality of life. First-line treatment with a vascular endothelial growth factor inhibitor remains the optimal approach in metastatic clear cell renal cell carcinom
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