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 Dissecting the Fine Details of Assembly of aT = 3 Phage Capsid

2005 ◽  
Vol 6 (2) ◽  
pp. 119-125 ◽  
Author(s):  
P. G. Stockley ◽  
A. E. Ashcroft ◽  
S. Francese ◽  
G. S. Thompson ◽  
N. A. Ranson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein Complexes ◽  
Model Systems ◽  
Replicase Gene ◽  
Nmr Studies ◽  
Partial Explanation ◽  
Stem Loop ◽  
Assembly Pathway

The RNA bacteriophages represent ideal model systems in which to probe the detailed assembly pathway for the formation of aT = 3 quasi-equivalent capsid. For MS2, the assembly reaction can be probedin vitrousing acid disassembled coat protein subunits and a short (19 nt) RNA stem-loop that acts as the translational operator of the replicase gene and leads to sequence-specific sequestration and packaging of the cognate phage RNAin vivo. Reassembly reactions can be initiated by mixing these components at neutral pH. The molecular basis of the sequence-specific RNA–protein interaction is now well understood. Recent NMR studies on the protein demonstrate extensive mobility in the loops of the polypeptide that alter their conformations to form the quasi-equivalent conformers of the final capsid. It seems reasonable to assume that RNA binding results in reduction of this flexibility. However, mass spectrometry suggests that these RNA–protein complexes may only provide one type of quasi-equivalent capsid building block competent to form five-fold axes but not the full shell. Work with longer RNAs suggests that the RNA may actively template the assembly pathway providing a partial explanation of how conformers are selected in the growing shell.

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 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

Conjugates of Classical DNA/RNA Binder with Nucleobase: Chemical, Biochemical and Biomedical Applications

2019 ◽  
Vol 26 (30) ◽  
pp. 5609-5624
Author(s):  
Dijana Saftić ◽  
Željka Ban ◽  
Josipa Matić ◽  
Lidija-Marija Tumirv ◽  
Ivo Piantanida
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Biomedical Applications ◽  
Protein Targets ◽  
New Class ◽  
Rna Targets ◽  
Covalently Linked ◽  
Structural Aspects

: Among the most intensively studied classes of small molecules (molecular weight < 650) in biomedical research are small molecules that non-covalently bind to DNA/RNA, and another intensively studied class is nucleobase derivatives. Both classes have been intensively elaborated in many books and reviews. However, conjugates consisting of DNA/RNA binder covalently linked to nucleobase are much less studied and have not been reviewed in the last two decades. Therefore, this review summarized reports on the design of classical DNA/RNA binder – nucleobase conjugates, as well as data about their interactions with various DNA or RNA targets, and even in some cases protein targets are involved. According to these data, the most important structural aspects of selective or even specific recognition between small molecule and target are proposed, and where possible related biochemical and biomedical aspects were discussed. The general conclusion is that this, rather new class of molecules showed an amazing set of recognition tools for numerous DNA or RNA targets in the last two decades, as well as few intriguing in vitro and in vivo selectivities. Several lead research lines show promising advancements toward either novel, highly selective markers or bioactive, potentially druggable molecules.

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 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.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

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