Publications by authors named "Andrei-Jose Petrescu"

29 Publications

  • Page 1 of 1

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network.

Mol Cell Proteomics 2021 Jul 29;20:100125. Epub 2021 Jul 29.

Department of Molecular Cell Biology, Institute of Biochemistry, Bucharest, Romania. Electronic address:

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mcpro.2021.100125DOI Listing
July 2021

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.

Int J Mol Sci 2021 Feb 22;22(4). Epub 2021 Feb 22.

Department of Molecular Cell Biology, Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.

EDEM3 recognizes and directs misfolded proteins to the ER-associated protein degradation (ERAD) process. EDEM3 was predicted to act as lectin or as a mannosidase because of its homology with the GH47 catalytic domain of the Man1B1, but the contribution of the other regions remained unresolved. Here, we dissect the molecular determinants governing EDEM3 function and its cellular interactions. LC/MS analysis indicates very few stable ER interactors, suggesting EDEM3 availability for transient substrate interactions. Sequence analysis reveals that EDEM3 consists of four consecutive modules defined as GH47, intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD) domain. Using an EDEM3 knock-out cell line, we expressed EDEM3 and domain deletion mutants to address EDEM3 function. We find that the mannosidase domain provides substrate binding even in the absence of mannose trimming and requires the IMD domain for folding. The PA and IDD domains deletions do not impair the trimming, but specifically modulate the turnover of two misfolded proteins, NHK and the soluble tyrosinase mutant. Hence, we demonstrate that EDEM3 provides a unique ERAD timing to misfolded glycoproteins, not only by its mannose trimming activity, but also by the positive and negative feedback modulated by the protease-associated and intrinsically disordered domain, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms22042172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7926307PMC
February 2021

Deep Learning in the Quest for Compound Nomination for Fighting COVID-19.

Curr Med Chem 2021 ;28(28):5699-5732

Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, Splaiul Independenţei 91-95, 050095 Bucharest, Romania.

The current COVID-19 pandemic initiated an unprecedented response from clinicians and the scientific community in all relevant biomedical fields. It created an incredible multidimensional data-rich framework in which deep learning proved instrumental to make sense of the data and build models used in prediction-validation workflows that in a matter of months have already produced results in assessing the spread of the outbreak, its taxonomy, population susceptibility, diagnostics or drug discovery and repurposing. More is expected to come in the near future by using such advanced machine learning techniques to combat this pandemic. This review aims to unravel just a small fraction of the large global endeavors by focusing on the research performed on the main COVID-19 targets, on the computational weaponry used in identifying drugs to combat the disease, and on some of the most important directions found to contain COVID-19 or alleviating its symptoms in the absence of specific medication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/0929867328666210113170222DOI Listing
September 2021

Robosample: A rigid-body molecular simulation program based on robot mechanics.

Biochim Biophys Acta Gen Subj 2020 08 13;1864(8):129616. Epub 2020 Apr 13.

Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, Bucharest 060031, Romania. Electronic address:

Background: Compared with all-atom molecular dynamics (MD), constrained MD methods allow for larger time steps, potentially reducing computational cost. For this reason, there has been continued interest in improving constrained MD algorithms to increase configuration space sampling in molecular simulations.

Methods: Here, we introduce Robosample, a software package that implements high-performance constrained dynamics algorithms, originally developed for robotics, and applies them to simulations of biomolecular systems. As in the gMolmodel package developed by Spiridon and Minh in 2017, Robosample uses Constrained Dynamics Hamiltonian Monte Carlo (CDHMC) as a Gibbs sampling move - a type of Monte Carlo move where a subset of coordinates is allowed to change. In addition to the previously described Cartesian and torsional dynamics moves, Robosample implements spherical and cylindrical joints that can be distributed along the molecule by the user.

Results: In alanine dipeptide simulations, the free energy surface is recovered by mixing fully flexible with torsional, cylindrical, or spherical dynamics moves. Ramachandran dynamics, where only the two key torsions are mobile, accelerate the slowest transition by an order of magnitude. We also show that simulations of a complex glycan cover significantly larger regions of the configuration space when mixed with constrained dynamics.

Major Conclusions: Robosample is a tool of choice for efficient conformational sampling of large biomolecules.

General Significance: Robosample is intended as a reliable and user-friendly simulation package for fast biomolecular sampling that does not require extensive expertise in mechanical engineering or in the statistical mechanics of reduced coordinates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2020.129616DOI Listing
August 2020

LRRpredictor-A New LRR Motif Detection Method for Irregular Motifs of Plant NLR Proteins Using an Ensemble of Classifiers.

Genes (Basel) 2020 03 8;11(3). Epub 2020 Mar 8.

Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 060031 Bucharest, Romania.

Leucine-rich-repeats (LRRs) belong to an archaic procaryal protein architecture that is widely involved in protein-protein interactions. In eukaryotes, LRR domains developed into key recognition modules in many innate immune receptor classes. Due to the high sequence variability imposed by recognition specificity, precise repeat delineation is often difficult especially in plant NOD-like Receptors (NLRs) notorious for showing far larger irregularities. To address this problem, we introduce here LRRpredictor, a method based on an ensemble of estimators designed to better identify LRR motifs in general but particularly adapted for handling more irregular LRR environments, thus allowing to compensate for the scarcity of structural data on NLR proteins. The extrapolation capacity tested on a set of annotated LRR domains from six immune receptor classes shows the ability of LRRpredictor to recover all previously defined specific motif consensuses and to extend the LRR motif coverage over annotated LRR domains. This analysis confirms the increased variability of LRR motifs in plant and vertebrate NLRs when compared to extracellular receptors, consistent with previous studies. Hence, LRRpredictor is able to provide novel insights into the diversification of LRR domains and a robust support for structure-informed analyses of LRRs in immune receptor functioning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes11030286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140858PMC
March 2020

Profiling Optimal Conditions for Capturing EDEM Proteins Complexes in Melanoma Using Mass Spectrometry.

Adv Exp Med Biol 2019 ;1140:155-167

Department of Molecular Cell Biology, Institute of Biochemistry, Bucharest, Romania.

Endoplasmic reticulum (ER) resident and secretory proteins that fail to reach their native conformation are selected for degradation through the ER-Associated Degradation (ERAD) pathway. The ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) were shown to be involved in this pathway but their precise role is still under investigation. Mass spectrometry analysis has contributed significantly to the characterization of protein complexes in the last years. The recent advancements in instrumentation, especially within resolution and speed can provide unique insights concerning the molecular architecture of protein-protein interactions in systems biology. Previous reports have suggested that several protein complexes in ERAD are sensitive to the extraction conditions. Indeed, whilst EDEM proteins can be recovered in most detergents, some of their partners are not solubilized, which further emphasizes the importance of the experimental setup. Here, we define such dynamic interactions of EDEM proteins by employing offline protein fractionation, nanoLC-MS/MS and describe how mass spectrometry can contribute to the characterization of such complexes, particularly within a disease context like melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-030-15950-4_9DOI Listing
September 2019

Genome-wide functional analyses of plant coiled-coil NLR-type pathogen receptors reveal essential roles of their N-terminal domain in oligomerization, networking, and immunity.

PLoS Biol 2018 12 12;16(12):e2005821. Epub 2018 Dec 12.

Molecular Plant Pathology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, the Netherlands.

The ability to induce a defense response after pathogen attack is a critical feature of the immune system of any organism. Nucleotide-binding leucine-rich repeat receptors (NLRs) are key players in this process and perceive the occurrence of nonself-activities or foreign molecules. In plants, coevolution with a variety of pests and pathogens has resulted in repertoires of several hundred diverse NLRs in single individuals and many more in populations as a whole. However, the mechanism by which defense signaling is triggered by these NLRs in plants is poorly understood. Here, we show that upon pathogen perception, NLRs use their N-terminal domains to transactivate other receptors. Their N-terminal domains homo- and heterodimerize, suggesting that plant NLRs oligomerize upon activation, similar to the vertebrate NLRs; however, consistent with their large number in plants, the complexes are highly heterometric. Also, in contrast to metazoan NLRs, the N-terminus, rather than their centrally located nucleotide-binding (NB) domain, can mediate initial partner selection. The highly redundant network of NLR interactions in plants is proposed to provide resilience to perturbation by pathogens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2005821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312357PMC
December 2018

Distinct Roles of Non-Overlapping Surface Regions of the Coiled-Coil Domain in the Potato Immune Receptor Rx1.

Plant Physiol 2018 11 7;178(3):1310-1331. Epub 2018 Sep 7.

Laboratory of Nematology, Department of Plant Sciences, Wageningen University, 6708 PB Wageningen, The Netherlands.

The intracellular immune receptor Rx1 of potato (), which confers effector-triggered immunity to , consists of a central nucleotide-binding domain (NB-ARC) flanked by a carboxyl-terminal leucine-rich repeat (LRR) domain and an amino-terminal coiled-coil (CC) domain. Rx1 activity is strictly regulated by interdomain interactions between the NB-ARC and LRR, but the contribution of the CC domain in regulating Rx1 activity or immune signaling is not fully understood. Therefore, we used a structure-informed approach to investigate the role of the CC domain in Rx1 functionality. Targeted mutagenesis of CC surface residues revealed separate regions required for the intramolecular and intermolecular interaction of the CC with the NB-ARC-LRR and the cofactor Ran GTPase-activating protein2 (RanGAP2), respectively. None of the mutant Rx1 proteins was constitutively active, indicating that the CC does not contribute to the autoinhibition of Rx1 activity. Instead, the CC domain acted as a modulator of downstream responses involved in effector-triggered immunity. Systematic disruption of the hydrophobic interface between the four helices of the CC enabled the uncoupling of cell death and disease resistance responses. Moreover, a strong dominant negative effect on Rx1-mediated resistance and cell death was observed upon coexpression of the CC alone with full-length Rx1 protein, which depended on the RanGAP2-binding surface of the CC. Surprisingly, coexpression of the N-terminal half of the CC enhanced Rx1-mediated resistance, which further indicated that the CC functions as a scaffold for downstream components involved in the modulation of disease resistance or cell death signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.18.00603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6236623PMC
November 2018

The Design of New HIV-IN Tethered Bifunctional Inhibitors Using Multiple Microdomain Targeted Docking.

Curr Med Chem 2019 ;26(15):2574-2600

Modern Catalysis Method Mihai Ciubotaru group, Latvian Institute of Organic Synthesis, Riga, Latvia.

Currently, used antiretroviral HIV therapy drugs exclusively target critical groups in the enzymes essential for the viral life cycle. Increased mutagenesis of their genes changes these viral enzymes, which once mutated can evade therapeutic targeting, effects which confer drug resistance. To circumvent this, our review addresses a strategy to design and derive HIV-Integrase (HIV-IN) inhibitors which simultaneously target two IN functional domains, rendering it inactive even if the enzyme accumulates many mutations. First we review the enzymatic role of IN to insert the copied viral DNA into a chromosome of the host T lymphocyte, highlighting its main functional and structural features to be subjected to inhibitory action. From a functional and structural perspective we present all classes of HIV-IN inhibitors with their most representative candidates. For each chosen compound we also explain its mechanism of IN inhibition. We use the recently resolved cryo EM IN tetramer intasome DNA complex onto which we dock various reference IN inhibitory chemical scaffolds such as to target adjacent functional IN domains. Pairing compounds with complementary activity, which dock in the vicinity of a IN structural microdomain, we design bifunctional new drugs which may not only be more resilient to IN mutations but also may be more potent inhibitors than their original counterparts. In the end of our review we propose synthesis pathways to link such paired compounds with enhanced synergistic IN inhibitory effects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/0929867325666180406114405DOI Listing
September 2019

Heavy metal accumulation by Saccharomyces cerevisiae cells armed with metal binding hexapeptides targeted to the inner face of the plasma membrane.

Appl Microbiol Biotechnol 2017 Jul 3;101(14):5749-5763. Epub 2017 Jun 3.

Faculty of Chemistry, University of Bucharest, Sos. Panduri 90-92, Bucharest, Romania.

Accumulation of heavy metals without developing toxicity symptoms is a phenotype restricted to a small group of plants called hyperaccumulators, whose metal-related characteristics suggested the high potential in biotechnologies such as bioremediation and bioextraction. In an attempt to extrapolate the heavy metal hyperaccumulating phenotype to yeast, we obtained Saccharomyces cerevisiae cells armed with non-natural metal-binding hexapeptides targeted to the inner face of the plasma membrane, expected to sequester the metal ions once they penetrated the cell. We describe the construction of S. cerevisiae strains overexpressing metal-binding hexapeptides (MeBHxP) fused to the carboxy-terminus of a myristoylated green fluorescent protein (myrGFP). Three non-toxic myrGFP-MeBHxP (myrGFP-H6, myrGFP-C6, and myrGFP-(DE)3) were investigated against an array of heavy metals in terms of their effect on S. cerevisiae growth, heavy metal (hyper) accumulation, and capacity to remove heavy metal from contaminated environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00253-017-8335-0DOI Listing
July 2017

Inhibition of N-glycan processing modulates the network of EDEM3 interactors.

Biochem Biophys Res Commun 2017 05 31;486(4):978-984. Epub 2017 Mar 31.

Departament of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, Splaiul Independenţei, nr. 296, 060031, Bucharest 17, Romania. Electronic address:

We present here data on EDEM3 network of ER resident interactors and the changes induced upon this network by perturbing the early ER N-glycan processing with mannosidase and glucosidase inhibitors. By coupling immunoprecipitation with mass spectrometry we identified EDEM3 interactors and assigned statistical significance to those most abundant ER-residents that might form functional complexes with EDEM3. We further show that this ER interaction network changes in both content and abundance upon treatment with kifunensine (kif) and N-butyldeoxynojirimycin (NB-DNJ) which suggests that when interfering with the N-glycan processing pathway, the functional complexes involving EDEM3 adapt to maintain the cellular homeostasis. In order to increase the scope of EDEM3 network contenders, the set of MS identified species was further supplemented with putative interactors derived from in silico simulations performed with STRING. Finally, the most interesting candidates to this network were further validated by immunoprecipitation coupled with Western Blotting, which strengthened the confidence in the inferred interactions. The data corroborated herein suggest that besides ER residents, EDEM3 interacts also with proteins involved in the ERAD cargo recognition and targeting to degradation translocation into the cytosol, including UBA1 and UBA2 ubiquitinating enzymes. In addition, the results indicate that this network of EDEM3 interactors is highly sensitive to interfering with early ER N-glycan processing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2017.03.143DOI Listing
May 2017

An LRR/Malectin Receptor-Like Kinase Mediates Resistance to Non-adapted and Adapted Powdery Mildew Fungi in Barley and Wheat.

Front Plant Sci 2016 15;7:1836. Epub 2016 Dec 15.

Pathogen-Stress Genomics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Stadt Seeland, Germany.

Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns released from microbial organisms. Here we describe (for ) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus f.sp. . Transgenic barley lines with silenced allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus f.sp. . Transient expression of the barley or wheat genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.01836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5156707PMC
December 2016

SPRYSEC Effectors: A Versatile Protein-Binding Platform to Disrupt Plant Innate Immunity.

Front Plant Sci 2016 20;7:1575. Epub 2016 Oct 20.

Laboratory of Nematology, Wageningen University Wageningen, Netherlands.

Persistent infections by sedentary plant-parasitic nematodes are a major threat to important food crops all over the world. These roundworms manipulate host plant cell morphology and physiology to establish sophisticated feeding structures. Key modifications to plant cells during their transition into feeding structures are largely attributed to the activity of effectors secreted by the nematodes. The SPRYSEC effectors were initially identified in the potato cyst nematodes and , and are characterized by a single SPRY domain, a non-catalytic domain present in modular proteins with different functions. The SPRY domain is wide-spread among eukaryotes and thought to be involved in mediating protein-protein interactions. Thus far, the SPRY domain is only reported as a functional domain in effectors of plant-parasitic nematodes, but not of other plant pathogens. SPRYSEC effectors have been implicated in both suppression and activation of plant immunity, but other possible roles in nematode virulence remain undefined. Here, we review the latest reports on the structure, function, and sequence diversity of SPRYSEC effectors, which provide support for a model featuring these effectors as a versatile protein-binding platform for the nematodes to target a wide range of host proteins during parasitism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fpls.2016.01575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071358PMC
October 2016

Cell death triggering and effector recognition by Sw-5 SD-CNL proteins from resistant and susceptible tomato isolines to Tomato spotted wilt virus.

Mol Plant Pathol 2016 12 14;17(9):1442-1454. Epub 2016 Aug 14.

Laboratory of Virology, Department of Plant Sciences, Wageningen University, Droevendaalsesteeg 1, Wageningen, PB, 6708, the Netherlands.

Only a limited number of dominant resistance genes acting against plant viruses have been cloned, and further functional studies of these have been almost entirely limited to the resistance genes Rx against Potato virus X (PVX) and N against Tobacco mosaic virus (TMV). Recently, the cell-to-cell movement protein (NS ) of Tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant (Avr) of Sw-5b-mediated resistance, a dominant resistance gene which belongs to the class of SD-CC-NB-LRR (Solanaceae domain-coiled coil-nucleotide-binding-leucine-rich repeat, SD-CNL) resistance genes. On transient expression of the NS protein in tomato and transgenic Nicotiana benthamiana harbouring the Sw-5b gene, a hypersensitive cell death response (HR) is triggered. Here, it is shown that high accumulation of the Sw-5b protein in N. benthamiana leaves, achieved by co-expression of the Sw-5b protein with RNA silencing suppressors (RSSs), leads to auto-activity in the absence of NS . In a similar approach, Sw-5a, the highest conserved paralogue of Sw-5b from Solanum peruvianum, also triggered HR by auto-activation, whereas the highest conserved orthologue from susceptible S. lycopersicum, named Sw-5a , did not. However, neither of the last two homologues was able to trigger an NS -dependent HR. Truncated and mutated versions of these Sw-5 proteins revealed that the NB-ARC [nucleotide-binding adaptor shared by Apaf-1 (from humans), R proteins and CED-4 (from nematodes)] domain is sufficient for the triggering of HR and seems to be suppressed by the SD-CC domain. Furthermore, a single mutation was sufficient to restore auto-activity within the NB-ARC domain of Sw-5a . When the latter domain was fused to the Sw-5b LRR domain, NS -dependent HR triggering was regained, but not in the presence of its own Sw-5a LRR domain. Expression analysis in planta revealed a nucleocytoplasmic localization pattern of Sw-5b, in which the SD-CC domain seems to be required for nuclear translocation. Although the Sw-5 N-terminal CC domain, in contrast with Rx, contains an additional SD, most findings from this study support a conserved role of domains within NB-LRR (NLR) proteins against plant viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mpp.12439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638320PMC
December 2016

Random mutagenesis of the nucleotide-binding domain of NRC1 (NB-LRR Required for Hypersensitive Response-Associated Cell Death-1), a downstream signalling nucleotide-binding, leucine-rich repeat (NB-LRR) protein, identifies gain-of-function mutations in the nucleotide-binding pocket.

New Phytol 2015 Oct 26;208(1):210-23. Epub 2015 May 26.

Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands.

Plant nucleotide-binding, leucine-rich repeat (NB-LRR) proteins confer immunity to pathogens possessing the corresponding avirulence proteins. Activation of NB-LRR proteins is often associated with induction of the hypersensitive response (HR), a form of programmed cell death. NRC1 (NB-LRR Required for HR-Associated Cell Death-1) is a tomato (Solanum lycopersicum) NB-LRR protein that participates in the signalling cascade leading to resistance to the pathogens Cladosporium fulvum and Verticillium dahliae. To identify mutations in NRC1 that cause increased signalling activity, we generated a random library of NRC1 variants mutated in their nucleotide-binding domain and screened them for the ability to induce an elicitor-independent HR in Nicotiana tabacum. Screening of 1920 clones retrieved 11 gain-of-function mutants, with 10 of them caused by a single amino acid substitution. All substitutions are located in or very close to highly conserved motifs within the nucleotide-binding domain, suggesting modulation of the signalling activity of NRC1. Three-dimensional modelling of the nucleotide-binding domain of NRC1 revealed that the targeted residues are centred around the bound nucleotide. Our mutational approach has generated a wide set of novel gain-of-function mutations in NRC1 and provides insight into how the activity of this NB-LRR is regulated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/nph.13459DOI Listing
October 2015

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes.

Mol Plant Microbe Interact 2014 Aug;27(8):835-45

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1094/MPMI-01-14-0009-RDOI Listing
August 2014

Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2.

Plant Physiol 2013 Jul 9;162(3):1510-28. Epub 2013 May 9.

Laboratory of Nematology, Department of Plant Sciences, Wageningen University, 6708 PB Wageningen, The Netherlands.

Many plant and animal immune receptors have a modular nucleotide-binding-leucine-rich repeat (NB-LRR) architecture in which a nucleotide-binding switch domain, NB-ARC, is tethered to a LRR sensor domain. The cooperation between the switch and sensor domains, which regulates the activation of these proteins, is poorly understood. Here, we report structural determinants governing the interaction between the NB-ARC and LRR in the highly homologous plant immune receptors Gpa2 and Rx1, which recognize the potato cyst nematode Globodera pallida and Potato virus X, respectively. Systematic shuffling of polymorphic sites between Gpa2 and Rx1 showed that a minimal region in the ARC2 and N-terminal repeats of the LRR domain coordinate the activation state of the protein. We identified two closely spaced amino acid residues in this region of the ARC2 (positions 401 and 403) that distinguish between autoactivation and effector-triggered activation. Furthermore, a highly acidic loop region in the ARC2 domain and basic patches in the N-terminal end of the LRR domain were demonstrated to be required for the physical interaction between the ARC2 and LRR. The NB-ARC and LRR domains dissociate upon effector-dependent activation, and the complementary-charged regions are predicted to mediate a fast reassociation, enabling multiple rounds of activation. Finally, we present a mechanistic model showing how the ARC2, NB, and N-terminal half of the LRR form a clamp, which regulates the dissociation and reassociation of the switch and sensor domains in NB-LRR proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1104/pp.113.218842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707565PMC
July 2013

Tyrosinase degradation is prevented when EDEM1 lacks the intrinsically disordered region.

PLoS One 2012 8;7(8):e42998. Epub 2012 Aug 8.

Department of Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Bucharest, Romania.

EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121-598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40-119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3414498PMC
February 2013

Ancient diversity of splicing motifs and protein surfaces in the wild emmer wheat (Triticum dicoccoides) LR10 coiled coil (CC) and leucine-rich repeat (LRR) domains.

Mol Plant Pathol 2012 Apr 23;13(3):276-87. Epub 2011 Sep 23.

Department of Evolutionary and Environmental Biology, Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel.

In this study, we explore the diversity and its distribution along the wheat leaf rust resistance protein LR10 three-dimensional structure. Lr10 is a leaf rust resistance gene encoding a coiled coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) class of protein. Lr10 was cloned and sequenced from 58 accessions representing diverse habitats of wild emmer wheat in Israel. Nucleotide diversity was very high relative to other wild emmer wheat genes (π= 0.029). The CC domain was found to be the most diverse domain and subject to positive selection. Superimposition of the diversity on the CC three-dimensional structure showed that some of the variable and positively selected residues were solvent exposed and may interact with other proteins. The LRR domain was relatively conserved, but showed a hotspot of amino acid variation between two haplotypes in the ninth repeat. This repeat was longer than the other LRRs, and three-dimensional modelling suggested that an extensive α helix structure was formed in this region. The two haplotypes also differed in splicing regulation motifs. In genotypes with one haplotype, an intron was alternatively spliced in this region, whereas, in genotypes with the other haplotype, this intron did not splice at all. The two haplotypes are proposed to be ancient and maintained by balancing selection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1364-3703.2011.00744.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638671PMC
April 2012

C-terminus glycans with critical functional role in the maturation of secretory glycoproteins.

PLoS One 2011 18;6(5):e19979. Epub 2011 May 18.

Department Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Bucharest, Romania.

The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs--one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019979PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3097235PMC
October 2011

Coiled-coil domain-dependent homodimerization of intracellular barley immune receptors defines a minimal functional module for triggering cell death.

Cell Host Microbe 2011 Mar;9(3):187-199

Department of Plant-Microbe Interactions, Max-Planck Institut für Pflanzenzüchtungsforschung, Carl-von-Linne Weg 10, 50829 Cologne, Germany. Electronic address:

Plants and animals have evolved structurally related innate immune sensors, designated NLRs, to detect intracellular nonself molecules. NLRs are modular, consisting of N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains, a central nucleotide-binding (NB) domain, and C-terminal leucine-rich repeats (LRRs). The polymorphic barley mildew A (MLA) locus encodes CC-containing allelic immune receptors recognizing effectors of the pathogenic powdery mildew fungus. We report the crystal structure of an MLA receptor's invariant CC domain, which reveals a rod-shaped homodimer. MLA receptors also self-associate in vivo, but self-association appears to be independent of effector-triggered receptor activation. MLA CC mutants that fail to self-interact impair in planta cell death activity triggered by the CC domain alone and by an autoactive full-length MLA receptor that mimics its ATP-bound state. Thus, CC domain-dependent dimerization of the immune sensor defines a minimal functional unit and implies a role for the dimeric CC module in downstream immune signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chom.2011.02.008DOI Listing
March 2011

Tyrosine 656 in topoisomerase IIβ is important for the catalytic activity of the enzyme: Identification based on artifactual +80-Da modification at this site.

Proteomics 2011 Mar 31;11(5):829-42. Epub 2011 Jan 31.

Clinical Pharmacology Program, Taussig Cancer Institute, Cleveland, OH, USA.

Topoisomerase (topo) II catalyzes topological changes in DNA. Although both human isozymes, topo IIα and β are phosphorylated, site-specific phosphorylation of topo IIβ is poorly characterized. Using LC-MS/MS analysis of topo IIβ, cleaved with trypsin, Arg C or cyanogen bromide (CNBr) plus trypsin, we detected four +80-Da modified sites: tyr656, ser1395, thr1426 and ser1545. Phosphorylation at ser1395, thr1426 and ser1545 was established based on neutral loss of H(3) PO(4) (-98 Da) in the CID spectra and on differences in 2-D-phosphopeptide maps of (32) P-labeled wild-type (WT) and S1395A or T1426A/S1545A mutant topo IIβ. However, phosphorylation at tyr656 could not be verified by 2-D-phosphopeptide mapping of (32) P-labeled WT and Y656F mutant protein or by Western blotting with phosphotyrosine-specific antibodies. Since the +80-Da modification on tyr656 was observed exclusively during cleavage with CNBr and trypsin, this modification likely represented bromination, which occurred during CNBr cleavage. Re-evaluation of the CID spectra identified +78/+80-Da fragment ions in CID spectra of two peptides containing tyr656 and tyr711, confirming bromination. Interestingly, mutation of only tyr656, but not ser1395, thr1326 or ser1545, decreased topo IIβ activity, suggesting a functional role for tyr656. These results, while identifying an important tyrosine in topo IIβ, underscore the importance of careful interpretation of modifications having the same nominal mass.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pmic.201000194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608106PMC
March 2011

Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains.

Plant Cell 2010 Dec 21;22(12):4195-215. Epub 2010 Dec 21.

Laboratory of Nematology, Department of Plant Sciences, Wageningen University, 6708 PB Wageningen, The Netherlands.

The Rx1 protein, as many resistance proteins of the nucleotide binding-leucine-rich repeat (NB-LRR) class, is predicted to be cytoplasmic because it lacks discernable nuclear targeting signals. Here, we demonstrate that Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus. The coiled coil (CC) domain was found to be required for accumulation of Rx1 in the nucleus, whereas the LRR domain promoted the localization in the cytoplasm. Analyses of structural subdomains of the CC domain revealed no autonomous signals responsible for active nuclear import. Fluorescence recovery after photobleaching and nuclear fractionation indicated that the CC domain binds transiently to large complexes in the nucleus. Disruption of the Rx1 resistance function and protein conformation by mutating the ATP binding phosphate binding loop in the NB domain, or by silencing the cochaperone SGT1, impaired the accumulation of Rx1 protein in the nucleus, while Rx1 versions lacking the LRR domain were not affected in this respect. Our results support a model in which interdomain interactions and folding states determine the nucleocytoplasmic distribution of Rx1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1105/tpc.110.077537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3027179PMC
December 2010

Interface analysis of the complex between ERK2 and PTP-SL.

PLoS One 2009 8;4(5):e5432. Epub 2009 May 8.

Department of Enzymology, Institute of Biochemistry, Bucharest, Romania.

The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2-PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0005432PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675061PMC
June 2009

Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis.

Mol Plant Pathol 2007 May;8(3):293-305

Laboratory of Nematology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, the Netherlands.

SUMMARY The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca(2+) is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1364-3703.2007.00394.xDOI Listing
May 2007

A Caenorhabditis elegans wild type defies the temperature-size rule owing to a single nucleotide polymorphism in tra-3.

PLoS Genet 2007 Mar 9;3(3):e34. Epub 2007 Jan 9.

Laboratory of Nematology, Wageningen University, Wageningen, The Netherlands.

Ectotherms rely for their body heat on surrounding temperatures. A key question in biology is why most ectotherms mature at a larger size at lower temperatures, a phenomenon known as the temperature-size rule. Since temperature affects virtually all processes in a living organism, current theories to explain this phenomenon are diverse and complex and assert often from opposing assumptions. Although widely studied, the molecular genetic control of the temperature-size rule is unknown. We found that the Caenorhabditis elegans wild-type N2 complied with the temperature-size rule, whereas wild-type CB4856 defied it. Using a candidate gene approach based on an N2 x CB4856 recombinant inbred panel in combination with mutant analysis, complementation, and transgenic studies, we show that a single nucleotide polymorphism in tra-3 leads to mutation F96L in the encoded calpain-like protease. This mutation attenuates the ability of CB4856 to grow larger at low temperature. Homology modelling predicts that F96L reduces TRA-3 activity by destabilizing the DII-A domain. The data show that size adaptation of ectotherms to temperature changes may be less complex than previously thought because a subtle wild-type polymorphism modulates the temperature responsiveness of body size. These findings provide a novel step toward the molecular understanding of the temperature-size rule, which has puzzled biologists for decades.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.0030034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1808073PMC
March 2007

An N-linked glycan modulates the interaction between the CD1d heavy chain and beta 2-microglobulin.

J Biol Chem 2006 Dec 27;281(52):40369-78. Epub 2006 Oct 27.

Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.

Human CD1d molecules consist of a transmembrane CD1 (cluster of differentiation 1) heavy chain in association with beta(2)-microglobulin (beta(2)m). Assembly occurs in the endoplasmic reticulum (ER) and involves the initial glycan-dependent association of the free heavy chain with calreticulin and calnexin and the thiol oxidoreductase ERp57. Folding and disulfide bond formation within the heavy chain occurs prior to beta(2)m binding. There are four N-linked glycans on the CD1d heavy chain, and we mutated them individually to ascertain their importance for the assembly and function of CD1d-beta(2)m heterodimers. None of the four were indispensable for assembly or the ability to bind alpha-galactosyl ceramide and to present it to human NKT cells. Nor were any required for the CD1d molecule to bind and present alpha-galactosyl ceramide after lysosomal processing of a precursor lipid, galactosyl-(alpha1-2)-galactosyl ceramide. However, one glycan, glycan 2 at Asn-42, proved to be of particular importance for the stability of the CD1d-beta(2)m heterodimer. A mutant CD1d heavy chain lacking glycan 2 assembled with beta(2)m and transported from the ER more rapidly than wild-type CD1d and dissociated more readily from beta(2)m upon exposure to detergents. A mutant expressing only glycan 1 dissociated completely from beta(2)m upon exposure to the detergent Triton X-100, whereas a mutant expressing only glycan 2 at Asn-42 was more stable. In addition, glycan 2 was not processed efficiently to the complex form in mature wild-type CD1d molecules. Modeling the glycans on the published structure indicated that glycan 2 interacts significantly with both the CD1d heavy chain and beta(2)m, which may explain these unusual properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M608518200DOI Listing
December 2006

Structural aspects of glycomes with a focus on N-glycosylation and glycoprotein folding.

Curr Opin Struct Biol 2006 Oct 30;16(5):600-7. Epub 2006 Aug 30.

Institute of Biochemistry, Splaiul Independenţei 296, 060031 Bucharest 17, Romania.

The pace of data accumulation in glycobiology has lately rapidly increased, largely due to high-throughput technologies. In this increasingly data-rich environment, computer science started to play a central role in handling the data, extracting significant biological information, and probing the missing parts of the 'scenery' by prediction, modelling or simulation. Investigating and comparing glycomes by bioinformatics and structural methods has great practical value and sharply increased in popularity in the past couple of years. In this context, advances have also been made with regard to structural aspects of protein N-glycosylation and consequences for glycoprotein folding. In these areas, however, an approach that integrates glycobiology with protein science is necessary.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2006.08.007DOI Listing
October 2006

Evaluation of a neural networks QSAR method based on ligand representation using substituent descriptors. Application to HIV-1 protease inhibitors.

J Mol Graph Model 2006 Sep 1;25(1):37-45. Epub 2005 Dec 1.

Institute of Biochemistry, Splaiul Independenţei 296, Sector 6, Bucharest, Romania.

We present here a neural networks method designed to predict biological activity based on a local representation of the ligand. The compounds of the series are represented by a vector mapping for each of four substituent properties: volume, log P, dipole moment and a simple 'steric' parameter relating to its shape. This ligand representation was tested using neural networks on a set of 42 cyclic-urea derivatives, inhibiting HIV-1 protease. The leave-one-out cross-validation using all descriptors in the input gave a correlation factor between prediction and experiment of 0.76 for the overall set and 0.88 when three outliers were left out. To rank the significance of the four descriptors, we further tested all combinations of two and three parameters for each substituent, using two disjunctive testing sets of five inhibitors. In these sets, vectors with extreme descriptor values were used either in the training or the testing set (sets A and B, respectively). The method is a very good interpolator (set A, 95+/-2% accuracy) but a less effective extrapolator (set B, 85+/-2% accuracy). Generally, the combinations including the 'steric' parameter predict better than average, while those containing the volume are less effective. The best prediction, 98.8+/-1.2%, was obtained when log P, the dipole and the steric parameter were used on set A. At the opposite end, the lowest ranked descriptor set was obtained when replacing log P with the volume, giving 92.3+/-6.7% accuracy over the set A.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmgm.2005.09.014DOI Listing
September 2006
-->