scholarly journals A Protein Engineering Approach for Uncovering Cryptic Ubiquitin-binding Sites: from a Ubiquitin-Variant Inhibitor of APC/C to K48 Chain Binding

2019 ◽  
Author(s):  
Edmond R. Watson ◽  
Christy R. R. Grace ◽  
Wei Zhang ◽  
Darcie J. Miller ◽  
Iain F. Davidson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Quality ◽  
Ex Vivo ◽  
Regulatory Proteins ◽  
Biological Processes ◽  
Anaphase Promoting Complex ◽  
C Terminus ◽  
Ubiquitin Binding ◽  
Enzymatic Cascades

ABSTRACTUbiquitin-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of ubiquitin regulation are conjugation through E1-E2-E3 enzymatic cascades, and recognition by ubiquitin-binding domains. An emerging theme in the ubiquitin field is that these two properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to ubiquitin’s C-terminus for ubiquitin transfer reactions, conjugation enzymes often bind non-covalently and weakly to ubiquitin at “exosites”. However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2 MDa E3 ligase Anaphase-Promoting Complex/Cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected ubiquitin-binding sites. Using a panel of ubiquitin variants (UbVs) we identify a protein-based inhibitor that blocks ubiquitin ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo EM structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a ubiquitin-binding exosite with preference for K48-linked chains. The results provide a new tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic ubiquitin binding sites within large multiprotein complexes.SIGNIFICANCE STATEMENTUbiquitin-mediated interactions influence numerous biological processes. These are often transient or a part of multivalent interactions. Therefore, unmasking these interactions remains a significant challenge for large, complicated enzymes such as the Anaphase-Promoting Complex/Cyclosome (APC/C), a multisubunit RING E3 ubiquitin (Ub) ligase. APC/C activity regulates numerous facets of biology by targeting key regulatory proteins for Ub-mediated degradation. Using a series of Ub variants (UbVs), we identified a new Ub-binding site on the APC/C that preferentially binds to K48-linked Ub chains. More broadly, we demonstrate a workflow that can be exploited to uncover Ub-binding sites within ubiquitylation machinery and other associated regulatory proteins to interrogate the complexity of the Ub code in biology.

scholarly journals Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site

2019 ◽  
Vol 116 (35) ◽  
pp. 17280-17289 ◽  
Author(s):  
Edmond R. Watson ◽  
Christy R. R. Grace ◽  
Wei Zhang ◽  
Darcie J. Miller ◽  
Iain F. Davidson ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Quality ◽  
Ex Vivo ◽  
Anaphase Promoting Complex ◽  
Ubiquitin Chain ◽  
Binding Domains ◽  
C Terminus ◽  
Enzymatic Cascades ◽  
Multistep Process

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub’s C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at “exosites.” However, identification of such sites is typically empirical and particularly challenging in large molecular machines. Here, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.

scholarly journals SiteOut: an online tool to design binding site-free DNA sequences

2015 ◽  
Author(s):  
Javier Estrada ◽  
Teresa Ruiz-Herrero ◽  
Clarissa Scholes ◽  
Zeba Wunderlich ◽  
Angela DePace
Keyword(s):  
Binding Site ◽  
Dna Sequences ◽  
Binding Sites ◽  
Regulatory Proteins ◽  
Biological Processes ◽  
Major Goal ◽  
Specific Sequence ◽  
Online Tool ◽  
Protein Binding Sites ◽  
Regulatory Dna

DNA-binding proteins control many fundamental biological processes such as transcription, recombination and replication. A major goal is to decipher the role that DNA sequence plays in orchestrating the binding and activity of such regulatory proteins. To address this goal, it is useful to rationally design DNA sequences with desired numbers, affinities and arrangements of protein binding sites. However, removing binding sites from DNA is computationally non-trivial since one risks creating new sites in the process of deleting or moving others. Here we present an online binding site removal tool, SiteOut, that enables users to design arbitrary DNA sequences that entirely lack binding sites for factors of interest. SiteOut can also be used to delete sites from a specific sequence, or to introduce site-free spacers between functional sequences without creating new sites at the junctions. In combination with commercial DNA synthesis services, SiteOut provides a powerful and flexible platform for synthetic projects that interrogate regulatory DNA. Here we describe the algorithm and illustrate the ways in which SiteOut can be used; it is publicly available at https://depace.med.harvard.edu/siteout/

scholarly journals Efficient Therapeutic Delivery by a Novel Cell-Penetrating Peptide Derived from Acinus

Cancers ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 1858
Author(s):  
Justine Habault ◽  
Claire Fraser ◽  
Ewa Pasquereau-Kotula ◽  
Maëlys Born-Bony ◽  
Anne Marie-Cardine ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Clinical Settings ◽  
Aspartic Acid Residue ◽  
Circulating Cells ◽  
C Terminus ◽  
Cell Penetrating ◽  
Endocytosis Pathway ◽  
Cancerous Cells

In this study, we have identified a novel cell-penetrating sequence, termed hAP10, from the C-terminus of the human protein Acinus. hAP10 was able to efficiently enter various normal and cancerous cells, likely through an endocytosis pathway, and to deliver an EGFP cargo to the cell interior. Cell penetration of a peptide, hAP10DR, derived from hAP10 by mutation of an aspartic acid residue to an arginine was dramatically increased. Interestingly, a peptide containing a portion of the heptad leucine repeat region domain of the survival protein AAC-11 (residues 377–399) fused to either hAP10 or hAP10DR was able to induce tumor cells, but not normal cells, death both ex vivo on Sézary patients’ circulating cells and to inhibit tumor growth in vivo in a sub-cutaneous xenograft mouse model for the Sézary syndrome. Combined, our results indicate that hAP10 and hAP10DR may represent promising vehicles for the in vitro or in vivo delivery of bioactive cargos, with potential use in clinical settings.

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

Identification of Three Tyrosine Phosphorylation Sites in the p53 Binding Domain of Apoptosis-Stimulating p53 Protein 2 (ASPP2) That Are Differentially Phosphorylated in Response to Chemotherapy in Patient Derived AML Blasts

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1396-1396
Author(s):  
Kerstin M Kampa-Schittenhelm ◽  
Charles D Lopez ◽  
Marcus M Schittenhelm
Keyword(s):  
Acute Leukemia ◽  
Tyrosine Phosphorylation ◽  
Ex Vivo ◽  
Phosphorylation Sites ◽  
Apoptosis Induction ◽  
P53 Mutations ◽  
Expression Levels ◽  
C Terminus ◽  
Response To Chemotherapy

Abstract Abstract 1396 Acute myeloid leukemias (AML) are difficult to treat, and risk-stratification for successful chemotherapy remains a major challenge. Inactivation of the p53 tumor suppressor pathway is a frequent event in many cancers that promotes tumorigenesis and resistance to chemotherapy. However, p53 mutations are rare in AML, and thus the p53-pathway must be inactivated by other mechanisms. ASPP2 is a haploinsufficient tumor suppressor that belongs to a family of p53-binding proteins that enhance apoptosis in part by stimulation of p53-transactivation of selected pro-apoptotic target genes. High ASPP2 expression levels in the absence of p53 mutations thereby argue for proper apoptosis induction capacity and thereby for better response rates. Indeed, low ASPP2 expression levels are correlated with aggressive courses of different tumors. As we have previously shown by qPCR (Kampa-Schittenhelm et al., ASH 2010) and confirm now by intracellular immunostaining in a larger patient cohort, ASPP2 expression levels vary widely in acute leukemias. In vitro silencing of ASPP2 transcription leads to abrogation of induction of apoptosis after application of chemotherapy, arguing for inferior in vivo response rates to therapy of patients lacking ASPP2 expression. Of note, the highest expression levels we have seen was in a patient with good prognosis core binding factor leukemia lacking an autoactivating KIT mutation. The p53 core domain must interact with the ASPP2 C-terminus to fully stimulate apoptotic function. To further investigate how regulation of the p53-ASPP2 interaction may play a role in apoptosis induction in AML, we identified several highly conserved and highly predicted tyrosine phosphorylation sites at the ASPP2 C-terminus. To study whether these sites modulate the p53-ASPP2 interaction and apoptotic function, we developed phospho-specific antibodies against the three highest-scoring phosphorylation sites and confirmed. tyrosine phosphorylation at Y1029, Y1046 and Y1114 in ex vivo blasts from AML patients. Intriguingly, based on the crystal structure of the p53-ASPP2 complex, phosphorylation of all three tyrosines is predicted to disrupt p53-ASPP2 binding. Tantalizingly, we found that these phosphorylation expression patterns changed after in vitro treatment of native blasts with chemotherapy: blasts treated with daunorubicin revealed an early change of tyrosine phosphorylation patterns. Using these new phospho-specific antibodies, we are continuing to analyze changes in phosphorylation patterns in primary AML blasts (with and without ex vivo chemotherapy) and are performing univariate and multivariate analysis to correlate with available clinical data. Preliminary data suggests that altered ASPP2 tyrosine phosphorylation in AML may play an important role in modulating response to chemotherapy-induced apoptosis in the absence of inactivating p53 mutations. Ongoing work is prospectively analyzing pY-ASPP2 in patients with acute leukemia during induction chemotherapy. These results aim to evaluate ASPP2 expression as an early-on prediction marker of therapy response in acute leukemia. Further, we aim to provide new and clinically relevant insight into p53 pathway inactivation in acute leukemia – which suggests a novel potential target for therapy to increase the effectiveness of chemotherapy in these patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Trypsin digestion of the inositol trisphosphate receptor: implications for the conformation and domain organization of the protein

1995 ◽  
Vol 307 (3) ◽  
pp. 859-865 ◽  
Author(s):  
S K Joseph ◽  
S Pierson ◽  
S Samanta
Keyword(s):  
Binding Sites ◽  
Time Course ◽  
Soluble Fraction ◽  
Phosphorylation Site ◽  
C Terminus ◽  
Proteolytic Site ◽  
Dependent Protein ◽  
Limited Digestion ◽  
Trisphosphate Receptor

Limited digestion of rat cerebellum microsomal vesicles with trypsin resulted in the proteolysis of the 240 kDa inositol 1,4,5-trisphosphate receptor (IP3R) and the formation of a 94 kDa species that remained membrane-bound and retained immunoreactivity to an antibody raised against the C-terminal sequence of this protein. The appearance of the 94 kDa species was associated with a loss of [3H]IP3 binding sites in the membrane and the appearance of [3H]IP3 binding sites in the soluble fraction. The 94 kDa fragment retained reactivity to biotinylated concanavalin A. In vitro phosphorylation of the IP3R in membranes with cyclic AMP-dependent protein kinase and [gamma-32P]ATP produced an unlabelled 94 kDa fragment after tryptic digestion. According to current models of the cerebellar IP3R this would place the proteolytic site between the phosphorylation site at serine-1755 and the first transmembrane segment of the IP3R. A second antibody raised to amino acids 401-414 in the N-terminal region of the receptor recognizes a 68 kDa fragment released into the soluble fraction after trypsin treatment. The time course of release of the 68 kDa fragment was correlated with the appearance of soluble binding sites, and the fragment was bound by IP3-Affigel resin. A large proportion of the 68 kDa fragment remained associated with the membrane fraction and could be specifically immunoprecipitated from detergent extracts of digested membranes by anti-C-terminus antibody. Our results provide experimental evidence to further localize the ligand binding domain and suggest that regions of the N-terminus and C-terminus may be non-covalently associated.

scholarly journals SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana

2015 ◽  
Vol 112 (35) ◽  
pp. 11108-11113 ◽  
Author(s):  
Ari Sadanandom ◽  
Éva Ádám ◽  
Beatriz Orosa ◽  
András Viczián ◽  
Cornelia Klose ◽  
...  
Keyword(s):  
Molecular Level ◽  
Expression Patterns ◽  
Red Light ◽  
Regulatory Proteins ◽  
Light Signaling ◽  
Phytochrome B ◽  
Sumo Proteases ◽  
C Terminus

The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyBLys996Arg-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases.

scholarly journals Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Joana S Ferreira ◽  
Thomas Papouin ◽  
Laurent Ladépêche ◽  
Andrea Yao ◽  
Valentin C Langlais ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ex Vivo ◽  
Membrane Dynamics ◽  
Relative Content ◽  
Receptor Trafficking ◽  
Glutamatergic Synapses ◽  
C Terminus ◽  
Hippocampal Synapses ◽  
Extracellular Microenvironment

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling.

scholarly journals Transcriptional Repression of the VC2105 Protein by Vibrio FadR Suggests that It Is a New Auxiliary Member of thefadRegulon

2016 ◽  
Vol 82 (9) ◽  
pp. 2819-2832 ◽  
Author(s):  
Rongsui Gao ◽  
Jingxia Lin ◽  
Han Zhang ◽  
Youjun Feng
Keyword(s):  
Transcriptional Regulation ◽  
Fatty Acid ◽  
Ligand Binding ◽  
Binding Site ◽  
Binding Sites ◽  
Regulatory Proteins ◽  
Content Type ◽  
Ligand Binding Sites

ABSTRACTRecently, our group along with others reported that theVibrioFadR regulatory protein is unusual in that, unlike the prototypicalfadRproduct ofEscherichia coli, which has only one ligand-binding site,VibrioFadR has two ligand-binding sites and represents a new mechanism for fatty acid sensing. The promoter region of thevc2105gene, encoding a putative thioesterase, was mapped, and a putative FadR-binding site (AA CTG GTA AGA GCA CTT) was proposed. Different versions of the FadR regulatory proteins were prepared and purified to homogeneity. Both electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) determined the direct interaction of thevc2105gene with FadR proteins of various origins. Further, EMSAs illustrated that the addition of long-chain acyl-coenzyme A (CoA) species efficiently dissociates thevc2105promoter from the FadR regulator. The expression level of theVibrio cholerae vc2105gene was elevated 2- to 3-fold in afadRnull mutant strain, validating that FadR is a repressor for thevc2105gene. The β-galactosidase activity of avc2105-lacZtranscriptional fusion was increased over 2-fold upon supplementation of growth medium with oleic acid. Unlike thefadDgene, a member of theVibrio fadregulon, the VC2105 protein played no role in bacterial growth and virulence-associated gene expression ofctxAB(cholera toxin A/B) andtcpA(toxin coregulated pilus A). Given that the transcriptional regulation ofvc2105fits the criteria for fatty acid degradation (fad) genes, we suggested that it is a new member of theVibrio fadregulon.IMPORTANCETheVibrioFadR regulator is unusual in that it has two ligand-binding sites. Different versions of the FadR regulatory proteins were prepared and characterizedin vitroandin vivo. An auxiliaryfadgene (vc2105) fromVibriowas proposed that encodes a putative thioesterase and has a predicted FadR-binding site (AAC TGG TA A GAG CAC TT). The function of this putative binding site was proved using both EMSA and SPR. Furtherin vitroandin vivoexperiments revealed that theVibrioFadR is a repressor for thevc2105gene. UnlikefadD, a member of theVibrio fadregulon, VC2105 played no role in bacterial growth and expression of the two virulence-associated genes (ctxABandtcpA). Therefore, since transcriptional regulation ofvc2105fits the criteria forfadgenes, it seems likely thatvc2105acts as a new auxiliary member of theVibrio fadregulon.

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