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1.
MAbs ; 16(1): 2341443, 2024.
Article in English | MEDLINE | ID: mdl-38666503

ABSTRACT

The development of bispecific antibodies that bind at least two different targets relies on bringing together multiple binding domains with different binding properties and biophysical characteristics to produce a drug-like therapeutic. These building blocks play an important role in the overall quality of the molecule and can influence many important aspects from potency and specificity to stability and half-life. Single-domain antibodies, particularly camelid-derived variable heavy domain of heavy chain (VHH) antibodies, are becoming an increasingly popular choice for bispecific construction due to their single-domain modularity, favorable biophysical properties, and potential to work in multiple antibody formats. Here, we review the use of VHH domains as building blocks in the construction of multispecific antibodies and the challenges in creating optimized molecules. In addition to exploring traditional approaches to VHH development, we review the integration of machine learning techniques at various stages of the process. Specifically, the utilization of machine learning for structural prediction, lead identification, lead optimization, and humanization of VHH antibodies.


Subject(s)
Antibodies, Bispecific , Machine Learning , Single-Domain Antibodies , Antibodies, Bispecific/immunology , Antibodies, Bispecific/chemistry , Humans , Single-Domain Antibodies/immunology , Single-Domain Antibodies/chemistry , Animals , Protein Engineering/methods , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/chemistry
2.
Bioconjug Chem ; 34(11): 2049-2054, 2023 11 15.
Article in English | MEDLINE | ID: mdl-37917829

ABSTRACT

Proteolysis targeting chimeras (PROTACs) are a family of heterobifunctional molecules that are now realizing their promise as a therapeutic strategy for targeted protein degradation. However, one limitation of existing designs is the lack of cell-selective targeting of the protein degrading payload. This manuscript reports a cell-targeted approach to degrade receptor-interacting serine/threonine-protein kinase 2 (RIPK2) in HER2+ cell lines. An antibody-PROTAC conjugate is prepared containing a protease-cleavable linkage between the antibody and the corresponding degrader. Potent RIPK2 degradation is observed in HER2+ cell lines, whereas an equivalent anti-IL4 antibody-PROTAC conjugate shows no degradation at therapeutically relevant concentrations. No RIPK2 degradation was observed in HER2- cell lines for both bioconjugates. This work demonstrates the potential for the cell-selective delivery of PROTAC scaffolds by engaging with signature extracellular proteins expressed on the surface of particular cell types.


Subject(s)
Immunoconjugates , Proteolysis Targeting Chimera , Cell Line , Proteolysis , Threonine , Serine , Ubiquitin-Protein Ligases
3.
Protein Expr Purif ; 198: 106125, 2022 10.
Article in English | MEDLINE | ID: mdl-35659600

ABSTRACT

The Type I Interferon cytokine family member, Interferon-α2b (hIFN-α2b), modulates a number of important biological mechanisms including anti-proliferation, immunoregulation and antiviral responses. Due to its role in the immune system, hIFN-α2b has been used as a therapeutic modulator in hepatitis C as well as some forms of leukaemia. Clinical grade hIFN-α2b is typically produced in bacterial expression systems that involves complex refolding protocols and subsequent loss of yields. In this study, we describe an expression and purification system for hIFN-α2b from mammalian cells. Application of the Trypsin-1 signal peptide-propeptide domain significantly improved the expression and secretion of hIFN-α2b from HEK293 cells. We established a simple purification strategy that yields homogenous, pure hIFN-α2b that is stable and biologically active.


Subject(s)
Interferon-alpha , Protein Sorting Signals , Animals , HEK293 Cells , Humans , Interferon alpha-2/genetics , Interferon-alpha/chemistry , Interferon-alpha/genetics , Mammals , Recombinant Proteins
4.
Biotechnol Lett ; 43(4): 757-765, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33415569

ABSTRACT

OBJECTIVE: To compare the effects of human Trypsin-1 signal peptide and pro-peptide on the expression and secretion efficiency of human Interleukin-25 from mammalian cells. RESULTS: The signal peptide and combined signal peptide-pro-peptide sequence of human Trypsin-1 improved the secretion of human IL-25 from 1.7 to 3.2 µg/ml and 1.7 to 8.2 µg/ml, respectively. Deletion analysis identified the minimal Trypsin-1 derived secretion domain that maintains improved human Interleukin-25 production and secretion. The presence of Trypsin-1 pro-peptide sequence does not affect the function of secreted human Interleukin-25. CONCLUSION: The Trypsin-1 signal peptide-pro-peptide sequence increased human IL-25 expression and secretion in mammalian cells by fivefold.


Subject(s)
Interleukin-17/genetics , Mutation , Protein Engineering/methods , Trypsin/chemistry , Gene Expression , HEK293 Cells , Humans , Interleukin-17/metabolism , Protein Domains , Protein Sorting Signals , Recombinant Proteins/metabolism , Trypsin/genetics
5.
MAbs ; 12(1): 1755069, 2020.
Article in English | MEDLINE | ID: mdl-32343620

ABSTRACT

Complex cellular targets such as G protein-coupled receptors (GPCRs), ion channels, and other multi-transmembrane proteins represent a significant challenge for therapeutic antibody discovery, primarily because of poor stability of the target protein upon extraction from cell membranes. To assess whether a limited set of membrane-bound antigen formats could be exploited to identify functional antibodies directed against such targets, we selected a GPCR of therapeutic relevance (CCR1) and identified target binders using an in vitro yeast-based antibody discovery platform (AdimabTM) to expedite hit identification. Initially, we compared two different biotinylated antigen formats overexpressing human CCR1 in a 'scouting' approach using a subset of the antibody library. Binders were isolated using streptavidin-coated beads, expressed as yeast supernatants, and screened using a high-throughput binding assay and flow cytometry on appropriate cell lines. The most suitable antigen was then selected to isolate target binders using the full library diversity. This approach identified a combined total of 183 mAbs with diverse heavy chain sequences. A subset of clones exhibited high potencies in primary cell chemotaxis assays, with IC50 values in the low nM/high pM range. To assess the feasibility of any further affinity enhancement, full-length hCCR1 protein was purified, complementary-determining region diversified libraries were constructed from a high and lower affinity mAb, and improved binders were isolated by fluorescence-activated cell sorting selections. A significant affinity enhancement was observed for the lower affinity parental mAb, but not the high affinity mAb. These data exemplify a methodology to generate potent human mAbs for challenging targets rapidly using whole cells as antigen and define a route to the identification of affinity-matured variants if required.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , Antibody Specificity/immunology , Receptors, CCR1/immunology , Receptors, G-Protein-Coupled/immunology , Animals , Antibodies, Monoclonal/pharmacology , CHO Cells , Cricetinae , Cricetulus , HEK293 Cells , Humans , Peptide Library , Protein Binding/drug effects , Receptors, CCR1/antagonists & inhibitors , Receptors, CCR1/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
6.
J Biol Chem ; 291(11): 5500-5511, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26728464

ABSTRACT

A potent VEGF inhibitor with novel antibody architecture and antigen binding mode has been developed. The molecule, hereafter referred to as VEGF dual dAb (domain antibody), was evaluated in vitro for binding to VEGF and for potency in VEGF-driven models and compared with other anti-VEGF biologics that have been used in ocular anti-angiogenic therapeutic regimes. VEGF dual dAb is more potent than bevacizumab and ranibizumab for VEGF binding, inhibition of VEGF receptor binding assays (RBAs), and VEGF-driven in vitro models of angiogenesis and displays comparable inhibition to aflibercept (Eylea). VEGF dual dAb is dimeric, and each monomer contains two distinct anti-VEGF domain antibodies attached via linkers to a human IgG1 Fc domain. Mechanistically, the enhanced in vitro potency of VEGF dual dAb, in comparison to other anti-VEGF biologics, can be explained by increased binding stoichiometry. A consistent model of the target engagement has been built based on the x-ray complexes of each of the two isolated domain antibodies with the VEGF antigen.


Subject(s)
Bevacizumab/pharmacology , Ranibizumab/pharmacology , Receptors, Vascular Endothelial Growth Factor/pharmacology , Recombinant Fusion Proteins/pharmacology , Single-Domain Antibodies/pharmacology , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Crystallography, X-Ray , Drug Discovery , HEK293 Cells , Humans , Models, Molecular , Single-Domain Antibodies/chemistry , Swine , Vascular Endothelial Growth Factor A/chemistry
7.
Nat Commun ; 5: 4857, 2014 Sep 11.
Article in English | MEDLINE | ID: mdl-25209408

ABSTRACT

The RhoGEF GEF-H1 can be sequestered in an inactive state on polymerized microtubules by the dynein motor light-chain Tctex-1. Phosphorylation of GEF-H1 Ser885 by PKA or PAK kinases creates an inhibitory 14-3-3-binding site. Here we show a new mode of GEF-H1 activation in response to the G-protein-coupled receptor (GPCR) ligands lysophosphatidic acid (LPA) or thrombin that is independent of microtubule depolymerization. LPA/thrombin stimulates disassembly of the GEF-H1:dynein multi-protein complex through the concerted action of Gα and Gßγ. Gα binds directly to GEF-H1 and displaces it from Tctex-1, while Gßγ binds to Tctex-1 and disrupts its interaction with the dynein intermediate chain, resulting in the release of GEF-H1. Full activation of GEF-H1 requires dephosphorylation of Ser885 by PP2A, which is induced by thrombin. The coordinated displacement of GEF-H1 from microtubules by G-proteins and its dephosphorylation by PP2A demonstrate a multistep GEF-H1 activation and present a unique mechanism coupling GPCR signalling to Rho activation.


Subject(s)
Dyneins/metabolism , Microtubules/metabolism , Receptors, G-Protein-Coupled/metabolism , Rho Guanine Nucleotide Exchange Factors/metabolism , 14-3-3 Proteins/metabolism , Animals , GTP-Binding Protein alpha Subunits, G12-G13/drug effects , GTP-Binding Protein alpha Subunits, G12-G13/metabolism , GTP-Binding Protein beta Subunits/drug effects , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/drug effects , GTP-Binding Protein gamma Subunits/metabolism , Lysophospholipids/pharmacology , Mice , Mice, Knockout , Phosphorylation , Receptors, G-Protein-Coupled/drug effects , Rho Guanine Nucleotide Exchange Factors/drug effects , Thrombin/pharmacology , p21-Activated Kinases/metabolism
8.
Cell Cycle ; 11(19): 3555-8, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-22895009

ABSTRACT

CEP192 is a centrosome protein that plays a critical role in centrosome biogenesis and function in mammals, Drosophila and C. elegans. Moreover, CEP192-depleted cells arrest in mitosis with disorganized microtubules, suggesting that CEP192's function in spindle assembly goes beyond its role in centrosome activity and pointing to a potentially more direct role in the regulation of the mitotic microtubule landscape. To better understand CEP192 function in mitosis, we used mass spectrometry to identify CEP192-interacting proteins. We previously reported that CEP192 interacts with NEDD1, a protein that associates with the γ-tubulin ring complex (γ-TuRC) and regulates its phosphorylation status during mitosis. Additionally, within the array of proteins that interact with CEP192, we identified the microtubule binding K63-deubiquitinase CYLD. Further analyses show that co-depletion of CYLD alleviates the bipolar spindle assembly defects observed in CEP192-depleted cells. This functional relationship exposes an intriguing role for CYLD in spindle formation and raises the tantalizing possibility that CEP192 promotes robust mitotic spindle assembly by regulating K63-polyubiquitin-mediated signaling through CYLD.


Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Lysine/metabolism , Spindle Apparatus/metabolism , Tumor Suppressor Proteins/metabolism , Deubiquitinating Enzyme CYLD , HEK293 Cells , HeLa Cells , Humans , Immunoprecipitation , Mass Spectrometry , Protein Binding
9.
Mol Cell Biol ; 32(17): 3585-93, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22751931

ABSTRACT

The binding of the eukaryotic initiation factor 4E (eIF4E) to the mRNA 5' cap structure is a rate-limiting step in mRNA translation initiation. eIF4E promotes ribosome recruitment to the mRNA. In Drosophila, the eIF4E homologous protein (d4EHP) forms a complex with binding partners to suppress the translation of distinct mRNAs by competing with eIF4E for binding the 5' cap structure. This repression mechanism is essential for the asymmetric distribution of proteins and normal embryonic development in Drosophila. In contrast, the physiological role of the mammalian 4EHP (m4EHP) was not known. In this study, we have identified the Grb10-interacting GYF protein 2 (GIGYF2) and the zinc finger protein 598 (ZNF598) as components of the m4EHP complex. GIGYF2 directly interacts with m4EHP, and this interaction is required for stabilization of both proteins. Disruption of the m4EHP-GIGYF2 complex leads to increased translation and perinatal lethality in mice. We propose a model by which the m4EHP-GIGYF2 complex represses translation of a subset of mRNAs during embryonic development, as was previously reported for d4EHP.


Subject(s)
Carrier Proteins/metabolism , Embryo, Mammalian/embryology , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , Protein Biosynthesis , Amino Acid Motifs , Animals , Carrier Proteins/chemistry , Embryo, Mammalian/metabolism , Eukaryotic Initiation Factor-4E/chemistry , Gene Expression Regulation, Developmental , HEK293 Cells , HeLa Cells , Humans , Mice , Mice, Knockout , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Maps
10.
Proteomics ; 12(10): 1576-90, 2012 May.
Article in English | MEDLINE | ID: mdl-22611051

ABSTRACT

Identifying the interactions established by a protein of interest can be a critical step in understanding its function. This is especially true when an unknown protein of interest is demonstrated to physically interact with proteins of known function. While many techniques have been developed to characterize protein-protein interactions, one strategy that has gained considerable momentum over the past decade for identification and quantification of protein-protein interactions, is affinity-purification followed by mass spectrometry (AP-MS). Here, we briefly review the basic principles used in affinity-purification coupled to mass spectrometry, with an emphasis on tools (both biochemical and computational), which enable the discovery and reporting of high quality protein-protein interactions.


Subject(s)
Chromatography, Affinity/methods , Mass Spectrometry/methods , Protein Interaction Mapping/methods , Proteins/chemistry , Proteins/metabolism , Animals , Humans , Proteins/analysis , Systems Biology/methods
11.
Cardiovasc Revasc Med ; 12(3): 184-186, 2011.
Article in English | MEDLINE | ID: mdl-21640938

ABSTRACT

First case of infective endocarditis on a percutaneous aortic valve replacement due to Moraxalla nonliquefaciens in 64 year old woman. It was successfully treated medically with antibiotics. She had not suitable for surgical aortic valve replacement due to 3 sternotomies for thymoma resection and subsequent radiotherapy with blocked major thoracic veins. Due to her azathioprine immunosuppresion (myasthenia) she may have been at increased endocarditis risk. We suggest prophylactic antibiotics at implant for this group in future.


Subject(s)
Aortic Valve Stenosis/therapy , Cardiac Catheterization/adverse effects , Endocarditis, Bacterial/microbiology , Heart Valve Prosthesis Implantation/adverse effects , Heart Valve Prosthesis/adverse effects , Moraxella/pathogenicity , Moraxellaceae Infections/microbiology , Prosthesis-Related Infections/microbiology , Anti-Bacterial Agents/therapeutic use , Azathioprine/adverse effects , Cardiac Catheterization/instrumentation , Endocarditis, Bacterial/drug therapy , Female , Heart Valve Prosthesis Implantation/instrumentation , Humans , Immunosuppressive Agents/adverse effects , Middle Aged , Moraxellaceae Infections/drug therapy , Myasthenia Gravis/drug therapy , Prosthesis-Related Infections/drug therapy , Risk Factors , Treatment Outcome
12.
Proteomics ; 11(8): 1508-16, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21360678

ABSTRACT

Affinity purification coupled to mass spectrometry (AP-MS) represents a powerful and proven approach for the analysis of protein-protein interactions. However, the detection of true interactions for proteins that are commonly considered background contaminants is currently a limitation of AP-MS. Here using spectral counts and the new statistical tool, Significance Analysis of INTeractome (SAINT), true interaction between the serine/threonine protein phosphatase 5 (PP5) and a chaperonin, heat shock protein 90 (Hsp90), is discerned. Furthermore, we report and validate a new interaction between PP5 and an Hsp90 adaptor protein, stress-induced phosphoprotein 1 (STIP1; HOP). Mutation of PP5, replacing key basic amino acids (K97A and R101A) in the tetratricopeptide repeat (TPR) region known to be necessary for the interactions with Hsp90, abolished both the known interaction of PP5 with cell division cycle 37 homolog and the novel interaction of PP5 with stress-induced phosphoprotein 1. Taken together, the results presented demonstrate the usefulness of label-free quantitative proteomics and statistical tools to discriminate between noise and true interactions, even for proteins normally considered as background contaminants.


Subject(s)
Nuclear Proteins/analysis , Phosphoprotein Phosphatases/analysis , Protein Interaction Mapping , Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Chaperonins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Humans , Mass Spectrometry , Mutation , Nuclear Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Binding , Proteomics
13.
Mol Cell ; 40(4): 619-31, 2010 Nov 24.
Article in English | MEDLINE | ID: mdl-21055983

ABSTRACT

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.


Subject(s)
DNA Replication , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Recombination, Genetic , Stress, Physiological , Cell Survival , DNA Breaks, Double-Stranded , HeLa Cells , Humans , NF-kappa B/chemistry , Protein Binding , S Phase , Templates, Genetic
14.
Atherosclerosis ; 212(1): 217-22, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20557887

ABSTRACT

OBJECTIVE: Chronic exenatide treatment in type 2 diabetes is associated with improved glucose control and fasting lipid levels, as well as weight loss. Less established is whether exenatide directly reduces postprandial lipid and lipoprotein levels without the reduction in body weight or fasting glucose and triglycerides levels that frequently occur with prolonged therapy. Therefore, the effect of a single injection of exenatide on postprandial lipids, remnant lipoproteins, and apolipoproteins was studied. METHODS: A double-blinded, randomized, placebo-controlled, crossover study was conducted in 35 subjects (31 men and 4 women) with impaired glucose tolerance (n=20) or recent onset type 2 diabetes (n=15). A single subcutaneous injection of exenatide (10 microg) or normal saline was administered just prior to a high-calorie, fat-enriched breakfast meal. Concentrations of triglycerides (TG), apolipoproteins B-48 and CIII, non-esterified fatty acids (NEFA), and remnant lipoprotein (RLP) cholesterol and TG in serum or plasma were measured prior to the injection and for up to 8 h postprandially. RESULTS: Exenatide markedly reduced postprandial elevation of TG, apolipoproteins B-48 and CIII, RLP-cholesterol and RLP-triglyceride (all p<0.001). Postprandial declines in NEFA were less pronounced but persisted longer with exenatide compared to placebo (p<0.05). These effects of exenatide were not affected either by glucose tolerance status or by treatment with statins. CONCLUSION: These results demonstrate that exenatide acutely and profoundly inhibits postprandial excursions of proatherogenic lipids and lipoproteins and may offer additional cardiovascular risk reduction (NCT00974272).


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glucose Intolerance/drug therapy , Hyperlipidemias/prevention & control , Hypoglycemic Agents/therapeutic use , Lipids/blood , Lipoproteins/blood , Peptides/therapeutic use , Venoms/therapeutic use , Adult , Aged , Apolipoprotein B-48/blood , Apolipoprotein C-III/blood , Arizona , Biomarkers/blood , Blood Glucose/drug effects , Blood Glucose/metabolism , Cholesterol/blood , Cross-Over Studies , Diabetes Mellitus, Type 2/blood , Dietary Fats/administration & dosage , Dietary Fats/metabolism , Double-Blind Method , Energy Intake , Exenatide , Fatty Acids, Nonesterified/blood , Female , Glucose Intolerance/blood , Humans , Hyperlipidemias/blood , Hypoglycemic Agents/administration & dosage , Injections, Subcutaneous , Insulin/blood , Male , Middle Aged , Peptides/administration & dosage , Postprandial Period , Time Factors , Treatment Outcome , Triglycerides/blood , Venoms/administration & dosage
15.
Diabetes Care ; 33(5): 1028-30, 2010 May.
Article in English | MEDLINE | ID: mdl-20200309

ABSTRACT

OBJECTIVE: Endothelial dysfunction is frequently present in individuals with insulin resistance or type 2 diabetes and can be induced by high-fat or high-carbohydrate meals. Because exenatide reduces postprandial glucose and lipid excursions, we hypothesized that it may also improve postprandial endothelial function. RESEARCH DESIGN AND METHODS: In a double-blinded randomized crossover design, postprandial endothelial function was examined in 28 individuals with impaired glucose tolerance or recent-onset type 2 diabetes after a single injection of exenatide or placebo given just before a high-fat meal. Endothelial function was determined with peripheral arterial tonometry pre- and postprandially. RESULTS: Postprandial endothelial function was higher after exenatide compared with placebo (P = 0.0002). In the placebo phase, postprandial change in endothelial function was inversely associated with mean postprandial concentrations of triglycerides (r = -0.62, P = 0.0004). Changes in postprandial triglyceride concentrations explained 64% of exenatide's effect on postprandial endothelial function. CONCLUSIONS: Exenatide ameliorates postprandial endothelial dysfunction after a high-fat meal.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glucose Intolerance/drug therapy , Hyperglycemia/drug therapy , Hypoglycemic Agents/administration & dosage , Peptides/administration & dosage , Venoms/administration & dosage , Adult , Age of Onset , Aged , Cohort Studies , Cross-Over Studies , Diabetes Mellitus, Type 2/metabolism , Dietary Fats/administration & dosage , Endothelium/drug effects , Endothelium/metabolism , Exenatide , Female , Glucose Intolerance/metabolism , Humans , Hyperglycemia/metabolism , Male , Middle Aged , Triglycerides/blood
16.
J Cardiovasc Med (Hagerstown) ; 11(1): 45-8, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19786890

ABSTRACT

Total anomalous venous drainage is a rare congenital cardiac defect seen in 1% of the population; there is usually a concomitant atrial septal defect. We discuss such a diagnostic challenge in a patient who presented on a number of occasions with acute respiratory distress and haemoptysis. True understanding of her underlying congenital heart disease was not appreciated until review in a congenital heart disease clinic - in which she presented for prepregnancy counselling. She was found to have a stricture at the site of anastomosis of the common chamber with the left atrium, resulting in pulmonary venous hypertension leading to acute presentation of pulmonary haemorrhages. She underwent successful full balloon dilatation of the site, leading to full recovery. Most patients are diagnosed at birth and undergo surgical repair. The outcome is good, and historically it has been regarded that it is unusual for them to have long-term complications, although dysrhythmias remain a recognized long-term complication. Surgery for total anomalous pulmonary venous drainage is nevertheless a palliation, and it is vital that the possible role of late complications is considered in all cases.


Subject(s)
Cardiac Surgical Procedures/adverse effects , Heart Defects, Congenital/surgery , Hypertension, Pulmonary/diagnosis , Pulmonary Circulation , Pulmonary Veins/surgery , Anemia, Iron-Deficiency/etiology , Anemia, Iron-Deficiency/physiopathology , Catheterization , Counseling , Diagnosis, Differential , Female , Heart Defects, Congenital/diagnosis , Heart Defects, Congenital/physiopathology , Hemoptysis/etiology , Hemoptysis/physiopathology , Humans , Hypertension, Pulmonary/etiology , Hypertension, Pulmonary/physiopathology , Hypertension, Pulmonary/therapy , Palliative Care , Preconception Care , Pregnancy , Pulmonary Veins/abnormalities , Pulmonary Veins/physiopathology , Respiratory Distress Syndrome/etiology , Respiratory Distress Syndrome/physiopathology , Treatment Outcome , Young Adult
17.
Curr Biol ; 19(10): 816-26, 2009 May 26.
Article in English | MEDLINE | ID: mdl-19427217

ABSTRACT

BACKGROUND: The assembly of a robust microtubule-based mitotic spindle is a prerequisite for the accurate segregation of chromosomes to progeny. Spindle assembly relies on the concerted action of centrosomes, spindle microtubules, molecular motors, and nonmotor spindle proteins. RESULTS: Here we use an RNA-interference screen of the human centrosome proteome to identify novel regulators of spindle assembly. One such regulator is HAUS, an 8-subunit protein complex that shares homology to Drosophila Augmin. HAUS localizes to interphase centrosomes and to mitotic spindle microtubules, and its disruption induces microtubule-dependent fragmentation of centrosomes along with an increase in centrosome size. HAUS disruption results in the destabilization of kinetochore microtubules and the eventual formation of multipolar spindles. These severe mitotic defects are alleviated by codepletion of NuMA, indicating that both factors regulate opposing activities. HAUS disruption alters NuMA localization, suggesting that mislocalized NuMA activity contributes to the spindle and centrosome defects observed. CONCLUSION: The human Augmin complex (HAUS) is a critical and evolutionary conserved multisubunit protein complex that regulates centrosome and spindle integrity.


Subject(s)
Centrosome/metabolism , Microtubule-Associated Proteins/metabolism , Multiprotein Complexes/metabolism , Protein Subunits/metabolism , Spindle Apparatus/metabolism , Animals , Antigens, Nuclear/genetics , Antigens, Nuclear/metabolism , Cell Cycle Proteins , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , HeLa Cells , Humans , Microtubule-Associated Proteins/genetics , Microtubules/metabolism , Nuclear Matrix-Associated Proteins/genetics , Nuclear Matrix-Associated Proteins/metabolism , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tubulin/genetics , Tubulin/metabolism
18.
J Immunol ; 182(6): 3837-45, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19265163

ABSTRACT

Neutrophil chemotaxis depends on actin dynamics, but the roles for specific cytoskeleton regulators in this response remain unclear. By analysis of mammalian diaphanous-related formin 1 (mDia1)-deficient mice, we have identified an essential role for this actin nucleator in neutrophil chemotaxis. Lack of mDia1 was associated with defects in chemoattractant-induced neutrophil actin polymerization, polarization, and directional migration, and also with impaired activation of RhoA, its downstream target p160-Rho-associated coil-containing protein kinase (ROCK), and the leukemia-associated RhoA guanine nucleotide exchange factor (LARG). Our data also revealed mDia1 to be associated with another cytoskeletal regulator, Wiskott-Aldrich syndrome protein (WASp), at the leading edge of chemotaxing neutrophils and revealed polarized morphology and chemotaxis to be more mildly impaired in WAS(-/-) than in mDia1(-/-) neutrophils, but essentially abrogated by combined mDia1/WASp deficiency. Thus, mDia1 roles in neutrophil chemotaxis appear to be subserved in concert with WASp and are realized at least in part by activation of the LARG/RhoA/ROCK signaling pathway.


Subject(s)
Carrier Proteins/physiology , Cell Polarity/immunology , Chemotaxis, Leukocyte/immunology , Guanine Nucleotide Exchange Factors/physiology , Neutrophils/immunology , Signal Transduction/immunology , rho GTP-Binding Proteins/physiology , rho-Associated Kinases/physiology , Animals , Carrier Proteins/genetics , Cell Movement/immunology , Feedback, Physiological/immunology , Fetal Proteins/deficiency , Fetal Proteins/genetics , Fetal Proteins/physiology , Formins , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins/deficiency , Microfilament Proteins/genetics , Microfilament Proteins/physiology , Neutrophils/cytology , Neutrophils/metabolism , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Rho Guanine Nucleotide Exchange Factors , Wiskott-Aldrich Syndrome Protein/physiology , rhoA GTP-Binding Protein
19.
Mol Cell Proteomics ; 8(1): 157-71, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18782753

ABSTRACT

The serine/threonine protein phosphatases are targeted to specific subcellular locations and substrates in part via interactions with a wide variety of regulatory proteins. Understanding these interactions is thus critical to understanding phosphatase function. Using an iterative affinity purification/mass spectrometry approach, we generated a high density interaction map surrounding the protein phosphatase 2A catalytic subunit. This approach recapitulated the assembly of the PP2A catalytic subunit into many different trimeric complexes but also revealed several new protein-protein interactions. Here we define a novel large multiprotein assembly, referred to as the striatin-interacting phosphatase and kinase (STRIPAK) complex. STRIPAK contains the PP2A catalytic (PP2Ac) and scaffolding (PP2A A) subunits, the striatins (PP2A regulatory B''' subunits), the striatin-associated protein Mob3, the novel proteins STRIP1 and STRIP2 (formerly FAM40A and FAM40B), the cerebral cavernous malformation 3 (CCM3) protein, and members of the germinal center kinase III family of Ste20 kinases. Although the function of the CCM3 protein is unknown, the CCM3 gene is mutated in familial cerebral cavernous malformations, a condition associated with seizures and strokes. Our proteomics survey indicates that a large portion of the CCM3 protein resides within the STRIPAK complex, opening the way for further studies of CCM3 biology. The STRIPAK assembly establishes mutually exclusive interactions with either the CTTNBP2 proteins (which interact with the cytoskeletal protein cortactin) or a second subcomplex consisting of the sarcolemmal membrane-associated protein (SLMAP) and the related coiled-coil proteins suppressor of IKKepsilon (SIKE) and FGFR1OP2. We have thus identified several novel PP2A-containing protein complexes, including a large assembly linking kinases and phosphatases to a gene mutated in human disease.


Subject(s)
Calmodulin-Binding Proteins/metabolism , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Multiprotein Complexes/metabolism , Nerve Tissue Proteins/metabolism , Phosphotransferases/metabolism , Protein Phosphatase 2/metabolism , Cytoskeletal Proteins , HeLa Cells , Humans , Phosphate-Binding Proteins , Protein Binding
20.
J Biol Chem ; 281(35): 25089-96, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16772297

ABSTRACT

This study explores the links between the GTPase RhoA and the serine kinase protein kinase D (PKD) during thymocyte development. The rationale is that RhoA and PKD regulate common biological responses during T cell development, but there is nothing known about their interdependence. In fibroblasts, Rho function is required for activation of PKD catalytic activity. However, the data show that activation of Rho is neither sufficient nor essential for PKD activation in T cells. One alternative explanation for the apparent convergence of PKD and Rho signaling in T cells is that PKD responses might be Rho-dependent. To address this latter possibility, we probed the Rho requirements for the actions of constitutively active PKD mutants in pre-T cells of transgenic mice. Active PKD can localize to either the plasma membrane or the cytosol, and we therefore compared the Rho requirements for the actions of membrane- or cytosol-localized PKD. Here we show that membrane-localized PKD regulation of pre-T cell differentiation is Rho-dependent, but the actions of cytosol-localized PKD are not. These studies demonstrate that a Rho requirement for PKD activation is not ubiquitous. Moreover, links between PKD and Rho are determined by the cellular location of PKD.


Subject(s)
Protein Kinase C/chemistry , rhoA GTP-Binding Protein/chemistry , rhoA GTP-Binding Protein/physiology , ADP Ribose Transferases/metabolism , Animals , Botulinum Toxins/metabolism , Cell Membrane/metabolism , Clostridium botulinum/metabolism , Cytosol/metabolism , Fibroblasts/metabolism , Humans , Jurkat Cells , Mice , Mice, Transgenic , Protein Kinase C/physiology , T-Lymphocytes/metabolism
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