scholarly journals Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes

2022 ◽  
Author(s):  
Benjamin J. Orlando ◽  
Pawel K. Dominik ◽  
Sourav Roy ◽  
Chinemerem Ogbu ◽  
Satchal K. Erramilli ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Protein Complexes ◽  
Gastrointestinal Diseases ◽  
Phage Display Library ◽  
Surface Receptors ◽  
Terminal Domain ◽  
A Cell ◽  
And Function ◽  
Development Structure

Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating 𝛽-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via 𝛽-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound 𝛽-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

scholarly journals Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Nicolas J. Delalez ◽  
Richard M. Berry ◽  
Judith P. Armitage
Keyword(s):  
Copy Number ◽  
Motor Proteins ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Content Type ◽  
Rotation Direction ◽  
Cell Measurement ◽  
Biological Complexes ◽  
And Function

ABSTRACTSome proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evidence is emerging that protein exchange can be part of an adaptive mechanism. The bacterial flagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direction of flagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, labeled with the fluorescent protein CyPet, inEscherichia coli. Our results confirm that,in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 ± 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 ± 17 and 144 ± 26 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by averaged fixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments.IMPORTANCEThe flagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch complex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adaptation at the motor.

scholarly journals Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.

1985 ◽  
Vol 5 (2) ◽  
pp. 342-351 ◽  
Author(s):  
J R Greenberg ◽  
E Carroll
Keyword(s):  
Mammalian Cells ◽  
Uv Light ◽  
Protein Complexes ◽  
Micrococcal Nuclease ◽  
Translation System ◽  
Cytoplasmic Process ◽  
Free Translation ◽  
Associated Proteins ◽  
A Cell ◽  
And Function

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.

A Cell-based Model of Hemostasis

2001 ◽  
Vol 85 (06) ◽  
pp. 958-965 ◽  
Author(s):  
Dougald Monroe ◽  
Maureane Hoffman
Keyword(s):  
Large Scale ◽  
Coagulation Factors ◽  
Cellular Receptors ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Coagulation Proteins ◽  
A Cell ◽  
And Control ◽  
Prevailing View

SummaryBased on our work and that of many other workers, we have developed a model of coagulation in vivo. Many workers have demonstrated mechanisms by which cells can influence the coagulation process. Nonetheless, the prevailing view of hemostasis remains that the protein coagulation factors direct and control the process with cells serving primarily to provide a phosphatidylserine containing surface on which the procoagulant complexes are assembled. By contrast, we propose a model in which coagulation is regulated by properties of cell surfaces. This model emphasizes the importance of specific cellular receptors for the coagulation proteins. Thus, cells with similar phosphatidylserine content can play very different roles in hemostasis depending on their complement of surface receptors. We propose that coagulation occurs not as a “cascade”, but in three overlapping stages: 1) initiation, which occurs on a tissue factor bearing cell; 2) amplification, in which platelets and cofactors are activated to set the stage for large scale thrombin generation; and 3) propagation, in which large amounts of thrombin are generated on the platelet surface. This cell based model explains some aspects of hemostasis that a protein-centric model does not.

scholarly journals The Structure and Function of Alkamides in Mammalian Systems

2021 ◽  
Author(s):  
Stephanie E. Johnstone ◽  
Scott M. Laster
Keyword(s):  
Fatty Acid ◽  
Intracellular Signaling ◽  
Viral Infections ◽  
Cellular Systems ◽  
Surface Receptors ◽  
Head Groups ◽  
Liver And Kidney ◽  
And Function

Alkamides, or alkylamides, are fatty acid amides produced by plants from the genera Echinacea, Acmella, Spilanthes, and Heliopsis among others. Alkamides contain varying head groups, an amide moiety, and a fatty acid tail with varying numbers of carbons and double and triple bonds. Extracts from these plants have been used worldwide by native peoples for the treatment of numerous medical disorders, including bacterial and viral infections, inflammation, liver and kidney disorders, and pain. In vitro, these molecules display a variety of different activities depending on the cell type tested. Studies with neurons, macrophages and mast cells have revealed interactions between alkamides and a number of different cells surface receptors and intracellular signaling molecules. Generally, the alkamides have been found to exert suppressive effects, inhibiting cellular activation. In this report we introduce the structure of alkamides and review their effects in a number of different cellular systems. We also describe structure:function studies that have been performed with alkamides. While these studies have not as yet revealed general rules for alkamide activity, interesting insights have been revealed. The stage is set for the development of synthetic, designer alkamides with targeted in vivo activities.

scholarly journals Functional partitioning of a liquid-like organelle during assembly of axonemal dyneins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Chanjae Lee ◽  
Rachael M Cox ◽  
Ophelia Papoulas ◽  
Amjad Horani ◽  
Kevin Drew ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Human Genetic Disease ◽  
Interaction Partners ◽  
A Cell ◽  
Cell Type Specific ◽  
Axonemal Dynein ◽  
And Function ◽  
Affinity Purification Mass Spectrometry ◽  
Functional Partitioning

Ciliary motility is driven by axonemal dyneins that are assembled in the cytoplasm before deployment to cilia. Motile ciliopathy can result from defects in the dyneins themselves or from defects in factors required for their cytoplasmic pre-assembly. Recent work demonstrates that axonemal dyneins, their specific assembly factors, and broadly-acting chaperones are concentrated in liquid-like organelles in the cytoplasm called DynAPs (Dynein Axonemal Particles). Here, we use in vivo imaging in Xenopus to show that inner dynein arm (IDA) and outer dynein arm (ODA) subunits are partitioned into non-overlapping sub-regions within DynAPs. Using affinity- purification mass-spectrometry of in vivo interaction partners, we also identify novel partners for inner and outer dynein arms. Among these, we identify C16orf71/Daap1 as a novel axonemal dynein regulator. Daap1 interacts with ODA subunits, localizes specifically to the cytoplasm, is enriched in DynAPs, and is required for the deployment of ODAs to axonemes. Our work reveals a new complexity in the structure and function of a cell-type specific liquid-like organelle that is directly relevant to human genetic disease.

scholarly journals Structural basis of altered potency and efficacy displayed by a major in vivo metabolite of the anti-diabetic PPARγ drug pioglitazone

2018 ◽  
Author(s):  
Sarah A. Mosure ◽  
Jinsai Shang ◽  
Richard Brust ◽  
Jie Zheng ◽  
Patrick R. Griffin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Activation Function ◽  
Structural Basis ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Exchange Mass Spectrometry ◽  
Pparγ Agonist ◽  
A Cell ◽  
And Function

ABSTRACTThe thiazolidinedione (TZD) pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes mellitus that binds and activates the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). Although TZDs have potent antidiabetic effects, they also display harmful side effects that have necessitated a better understanding of their mechanisms of action. In particular, little is known about the effect of in vivo TZD metabolites on the structure and function of PPARγ. Here, we present a structure-function comparison of Pio and a major in vivo metabolite, 1-hydroxypioglitazone (PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays compared to Pio. To determine the structural basis of these findings, we solved an X-ray crystal structure of PioOH bound to PPARγ ligand-binding domain (LBD) and compared it to a published Pio-bound crystal structure. PioOH exhibited an altered hydrogen bonding network that could underlie its reduced affinity and potency compared to Pio. Solution-state structural analysis using NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PioOH stabilizes the PPARγ activation function-2 (AF-2) coactivator binding surface better than Pio. In support of AF-2 stabilization, PioOH displayed stabilized coactivator binding in biochemical assays and better transcriptional efficacy (maximal transactivation response) in a cell-based assay that reports on the activity of the PPARγ LBD. These results, which indicate that Pio hydroxylation affects both its potency and efficacy as a PPARγ agonist, contribute to our understanding of PPARγ-binding drug metabolite interactions and may assist in future PPARγ drug design efforts.

scholarly journals Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

2021 ◽  
Author(s):  
Tal Noy-Porat ◽  
Adva Mechaly ◽  
Yinon Levy ◽  
Efi Makdasi ◽  
Ron Alcalay ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Phage Display Library ◽  
Host Cells ◽  
Terminal Domain ◽  
Isolation And Characterization ◽  
Recent Emergence ◽  
Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

scholarly journals Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

2014 ◽  
Vol 204 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Alex J. Smith ◽  
Byung-Ju Jin ◽  
Julien Ratelade ◽  
Alan S. Verkman
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Water Channel ◽  
Aquaporin 4 ◽  
Cellular Functions ◽  
Aggregation State ◽  
Functional Roles ◽  
State Dependent ◽  
And Function

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.

scholarly journals Neuroimmunoendocrine Regulation of the Prion Protein in Neutrophils

2012 ◽  
Vol 287 (42) ◽  
pp. 35506-35515 ◽  
Author(s):  
Rafael M. Mariante ◽  
Alberto Nóbrega ◽  
Rodrigo A. P. Martins ◽  
Rômulo B. Areal ◽  
Maria Bellio ◽  
...  
Keyword(s):  
Prion Protein ◽  
Surface Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathies ◽  
Systemic Injection ◽  
A Cell ◽  
And Function ◽  
Acute And Chronic Inflammation ◽  
The Impact

The prion protein (PrPC) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrPC may be implicated in other degenerative conditions often associated with inflammation. PrPC is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrPC in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrPC in mouse neutrophils. Up-regulation of PrPC was dependent on the serum content of TGF-β and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-β, either alone or in combination, directly up-regulated PrPC in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrPC. Moreover, GC also mediated up-regulation of PrPC in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrPC presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrPC in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.

scholarly journals Reconstitution of Two Recombinant LSm Protein Complexes Reveals Aspects of Their Architecture, Assembly, and Function

2005 ◽  
Vol 280 (16) ◽  
pp. 16066-16075 ◽  
Author(s):  
Bozidarka Zaric ◽  
Mohamed Chami ◽  
Hervé Rémigy ◽  
Andreas Engel ◽  
Kurt Ballmer-Hofer ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Cellular Transport ◽  
Core Domain ◽  
Assembly Pathway ◽  
Rna Targets ◽  
Transport Assay ◽  
Form Ring ◽  
And Function

Sm and Sm-like (LSm) proteins form complexes engaging in various RNA-processing events. Composition and architecture of the complexes determine their intracellular distribution, RNA targets, and function. We have reconstituted the human LSm1–7 and LSm2–8 complexes from their constituent componentsin vitro. Based on the assembly pathway of the canonical Sm core domain, we used heterodimeric and heterotrimeric sub-complexes to assemble LSm1–7 and LSm2–8. Isolated sub-complexes form ring-like higher order structures. LSm1–7 is assembled and stable in the absence of RNA. LSm1–7 forms ring-like structures very similar to LSm2–8 at the EM level. Ourin vitroreconstitution results illustrate likely features of the LSm complex assembly pathway. We prove the complexes to be functional both in an RNA bandshift and anin vivocellular transport assay.

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