scholarly journals Unraveling the mechanism of the cadherin-catenin-actin catch bond

2018 ◽  
Author(s):  
Shishir Adhikari ◽  
Jacob Moran ◽  
Christopher Weddle ◽  
Michael Hinczewski
Keyword(s):  
Protein Complexes ◽  
Optical Tweezer ◽  
Force Transducer ◽  
Salt Bridges ◽  
Lifetime Distributions ◽  
Catch Bond ◽  
The Individual ◽  
And Function

The adherens junctions between epithelial cells involve a protein complex formed by E-cadherin, β-catenin, α-catenin and F-actin. The stability of this complex was a puzzle for many years, since in vitro studies could reconstitute various stable subsets of the individual proteins, but never the entirety. The missing ingredient turned out to be mechanical tension: a recent experiment that applied physiological forces to the complex with an optical tweezer dramatically increased its lifetime, a phenomenon known as catch bonding. However, in the absence of a crystal structure for the full complex, the microscopic details of the catch bond mechanism remain mysterious. Building on structural clues that point to α-catenin as the force transducer, we present a quantitative theoretical model for how the catch bond arises, fully accounting for the experimental lifetime distributions. The model allows us to predict the energetic changes induced by tension at the interface between α-catenin and F-actin. It also identifies a significant energy barrier due to a network of salt bridges between two conformational states of β-catenin. By stabilizing one of these states, this barrier could play a role in how the complex responds to additional in vivo binding partners like vinculin. Since significant conformational energy barriers are a common feature of other adhesion systems that exhibit catch bonds, our model can be adapted into a general theoretical framework for integrating structure and function in a variety of force-regulated protein complexes.

scholarly journals Posttranslational Modifications and the Immunogenicity of Biotherapeutics

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Roy Jefferis
Keyword(s):  
Posttranslational Modifications ◽  
Drug Product ◽  
Molecular Forms ◽  
Phagocytic Cells ◽  
Production Platform ◽  
The Individual ◽  
And Function ◽  
Antigen Presenting

Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) bothin vivoandin vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.

scholarly journals The Lyt-2 molecule recognizes residues in the class I alpha 3 domain in allogeneic cytotoxic T cell responses.

1988 ◽  
Vol 168 (1) ◽  
pp. 325-341 ◽  
Author(s):  
J M Connolly ◽  
T A Potter ◽  
E M Wormstall ◽  
T H Hansen
Keyword(s):  
Primary Response ◽  
Class I ◽  
Single Amino Acid ◽  
Cytotoxic T Cell ◽  
Mhc Class I Molecule ◽  
Cytotoxic T Cell Responses ◽  
The Individual ◽  
And Function

The involvement of the different domains of the MHC class I molecule in CTL recognition was investigated. mAbs specific for the alpha 1/alpha 2 domains of H-2Ld interfered with both the primary and secondary generation and effector function of in vitro Ld-specific CTL. mAbs specific for the alpha 3 domain of H-2Ld interfered with the generation and function of primary in vitro Ld-specific CTL; however, there was no effect on the in vitro generation of secondary CTL and only partial inhibition of their function. In vivo treatment with graft-specific antibodies to both the alpha 3 domain and the alpha 1/alpha 2 domains together resulted in a dramatic enhancement of Ld- or Dd-disparate skin grafts, whereas the individual mAbs showed minimal effects. This suggested that the class I alpha 3 domain is recognized by alloreactive CTL. Several approaches were undertaken to examine whether recognition of the alpha 3 domain determinants is mediated by the Lyt-2 molecule. When mAbs specific for the alpha 3 domain of either H-2Ld or H-2Dd were used in vivo and in vitro, the resulting CTL population was not inhibited by antibody to the alpha 3 domain and was only partially inhibited by antibody to Lyt-2. We therefore observed a correlation between the effects of antibody to the class I alpha 3 domain of the target molecule and antibody to the Lyt-2 molecule on the CTL. To further test the relationship between CTL recognition of the alpha 3 domain and the involvement of Lyt-2, we used a cell expressing a mutation in the alpha 3 domain of the Dd molecule. The mutation resulted in a single amino acid substitution of glu to lys at residue 227 of the alpha 3 domain. Consistent with an earlier report, cells expressing the mutant Dd lys molecule were not lysed by CTL from a primary stimulation against the wild-type Dd glu molecule. However, this same cell line was killed by the Lyt-2-independent secondary Dd-specific CTL generated in the presence of antibody to the alpha 3 domain in vivo and in vitro. Furthermore, cells expressing the mutant Dd lys molecule failed to stimulate a primary response. In conclusion, several independent lines of evidence indicate that residues in the alpha 3 domain of the class I molecule are involved in recognition by the Lyt-2 molecule, and that Lyt-2-mediated recognition can be specifically blocked using mAb to determinants in the alpha 3 domain.

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.

scholarly journals Two novel heteropolymer-forming proteins maintain multicellular shape of the cyanobacteriumAnabaenasp. PCC 7120

2019 ◽  
Author(s):  
Benjamin L. Springstein ◽  
Dennis J. Nürnberg ◽  
Christian Woehle ◽  
Julia Weissenbach ◽  
Marius L. Theune ◽  
...  
Keyword(s):  
Double Mutant ◽  
Protein Complexes ◽  
Coiled Coil ◽  
Filamentous Structure ◽  
Cellular Processes ◽  
Junction Protein ◽  
And Function ◽  
Pcc 7120

AbstractPolymerizing and filament-forming proteins are instrumental for numerous cellular processes such as cell division and growth. Their function in stabilization and localization of protein complexes and replicons is achieved by a filamentous structure. Known filamentous proteins assemble into homopolymers consisting of single subunits – e.g. MreB and FtsZ in bacteria – or heteropolymers that are composed of two subunits, e.g. keratin and α/β tubulin in eukaryotes. Here, we describe two novel coiled-coil-rich proteins (CCRPs) in the filament forming cyanobacteriumAnabaenasp. PCC 7120 (hereafterAnabaena) that assemble into a heteropolymer and function in the maintenance of theAnabaenamulticellular shape (termed trichome). The two CCRPs – Alr4504 and Alr4505 (named ZicK and ZacK) – are strictly interdependent for the assembly of protein filamentsin vivoand polymerize nucleotide-independentlyin vitro, similar to known intermediate filament (IF) proteins. A ΔzicKΔzacK double mutant is characterized by a zigzagged cell arrangement and hence a loss of the typical linearAnabaenatrichome shape. ZicK and ZacK interact with themselves, with each other, with the elongasome protein MreB, the septal junction protein SepJ and the divisome associate septal protein SepI. Our results suggest that ZicK and ZacK function in cooperation with SepJ and MreB to stabilize theAnabaenatrichome and are likely essential for the manifestation of the multicellular shape inAnabaena. Our study reveals the presence of filament-forming IF-like proteins whose function is achieved through the formation of heteropolymers in cyanobacteria.

Photoinhibition in vivo and in vitro Involves Weakly Coupled Chlorophyll–Protein Complexes‡¶

2002 ◽  
Vol 75 (6) ◽  
pp. 613 ◽  
Author(s):  
Stefano Santabarbara ◽  
Ilaria Cazzalini ◽  
Andrea Rivadossi ◽  
Flavio M. Garlaschi ◽  
Giuseppe Zucchelli ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Weakly Coupled ◽  
Chlorophyll Protein Complexes ◽  
Chlorophyll Protein

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4221
Author(s):  
Aage Kristian Olsen Alstrup ◽  
Svend Borup Jensen ◽  
Ole Lerberg Nielsen ◽  
Lars Jødal ◽  
Pia Afzelius
Keyword(s):  
Animal Model ◽  
Animal Models ◽  
Porcine Model ◽  
Animal Experiments ◽  
Radioactive Tracers ◽  
The Individual ◽  
Selection Of

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

scholarly journals Extracellular vesicles as mediators and markers of acute organ injury: current concepts

2021 ◽  
Author(s):  
Birte Weber ◽  
Niklas Franz ◽  
Ingo Marzi ◽  
Dirk Henrich ◽  
Liudmila Leppik
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Organ Injury ◽  
Isolation And Characterization ◽  
Communication Processes ◽  
Incidence And Mortality ◽  
New Biomarkers ◽  
And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

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