scholarly journals Cytosolic delivery of large supramolecular protein complexes arranged on DNA nanopegboards

2017 ◽  
Author(s):  
Pavel M. Nikolov ◽  
Katja J. Koßmann ◽  
Alessa Schilling ◽  
Alessandro Angelin ◽  
Josipa Brglez ◽  
...  
Keyword(s):  
Cell Biology ◽  
Glucose Sensor ◽  
Protein Complexes ◽  
Cell Signalling ◽  
Model Systems ◽  
Dna Nanostructures ◽  
Multifunctional Protein ◽  
Multiprotein Complexes ◽  
Intracellular Targeting ◽  
Cytosolic Delivery

AbstractA generic methodology for cytosolic delivery of large supramolecular multiprotein complexes into living cells is described that takes advantage of the highly-controllable bottom-up fabrication of protein-decorated DNA nanostructures and the microfluidic “cell squeezing” technique. Therein, cells are deformed upon passage through a narrow constriction leading to formation of transient holes in the cell membrane that enable the diffusion of the protein-DNA nanostructures from the surrounding buffer into the cytosol. A diverse set of multiprotein complexes was assembled on DNA origami nanostructures using streptavidin and the sensitive glucose sensor protein FLIP as model systems. We demonstrate that our approach allows for the direct cytosolic delivery of these multifunctional protein complexes into the cytosol of HeLa cells. We also demonstrate that targeting groups can be incorporated into the protein-DNA nanoassemblies to enable their intracellular targeting to cytosolic compartments, such as the cytoskeleton or nucleus. We believe that this methodology will open up novel strategies for research in fundamental cell biology, such as the reverseengineering of the supramolecular machinery involved in gene regulation, cell signalling, or cell division. Furthermore, direct applications in immunotherapy can be foreseen.

scholarly journals Pseudo-DUBs as allosteric activators and molecular scaffolds of protein complexes

2018 ◽  
Vol 46 (2) ◽  
pp. 453-466 ◽  
Author(s):  
Miriam Walden ◽  
Safi Kani Masandi ◽  
Krzysztof Pawłowski ◽  
Elton Zeqiraj
Keyword(s):  
Cell Biology ◽  
Protein Complexes ◽  
Cell Signalling ◽  
Control Cell ◽  
Mechanisms Of Action ◽  
Molecular Scaffolds ◽  
Polyubiquitin Chains ◽  
Macromolecular Complexes ◽  
Cellular Life ◽  
Almost All

The ubiquitin (Ub) proteasome system and Ub signalling networks are crucial to cell biology and disease development. Deubiquitylases (DUBs) control cell signalling by removing mono-Ub and polyubiquitin chains from substrates. DUBs take part in almost all processes that regulate cellular life and are frequently dysregulated in disease. We have catalogued 99 currently known DUBs in the human genome and sequence conservation analyses of catalytic residues suggest that 11 lack enzyme activity and are classed as pseudo-DUBs. These pseudoenzymes play important biological roles by allosterically activating catalytically competent DUBs as well as other active enzymes. Additionally, pseudoenzymes act as assembly scaffolds of macromolecular complexes. We discuss how pseudo-DUBs have lost their catalytic activity, their diverse mechanisms of action and their potential as therapeutic targets. Many known pseudo-DUBs play crucial roles in cell biology and it is likely that unstudied and overlooked pseudo-DUB genes will have equally important functions.

Biophysics (and sociology) of ceramides.

2005 ◽  
Vol 72 ◽  
pp. 177-188 ◽  
Author(s):  
Félix M. Goñi ◽  
F-Xabier Contreras ◽  
L-Ruth Montes ◽  
Jesús Sot ◽  
Alicia Alonso
Keyword(s):  
Cell Biology ◽  
Positive Curvature ◽  
Acyl Chain ◽  
Cell Signalling ◽  
Hexagonal Structure ◽  
Lipid Monolayer ◽  
Flip Flop ◽  
Long Chain ◽  
Bilayer Permeability

In the past decade, the long-neglected ceramides (N-acylsphingosines) have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell signalling). Most natural ceramides have a long (16-24 C atoms) N-acyl chain, but short N-acyl chain ceramides (two to six C atoms) also exist in Nature, apart from being extensively used in experimentation, because they can be dispersed easily in water. Long-chain ceramides are among the most hydrophobic molecules in Nature, they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. In situ enzymic generation, or external addition, of long-chain ceramides in membranes has at least three important effects: (i) the lipid monolayer tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure, is increased, (ii) bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) transbilayer (flip-flop) lipid motion is promoted. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or non-existent.

scholarly journals X-ray and UV Radiation Damage of dsDNA/Protein Complexes

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3132
Author(s):  
Paweł Wityk ◽  
Dorota Kostrzewa-Nowak ◽  
Beata Krawczyk ◽  
Michał Michalik ◽  
Robert Nowak
Keyword(s):  
Dna Damage ◽  
Simple Model ◽  
Uv Radiation ◽  
Protein Complexes ◽  
Chemical Processes ◽  
Model Systems ◽  
Degradation Pathways ◽  
X Ray ◽  
Physico Chemical ◽  
Sensitization Process

Radiation and photodynamic therapies are used for cancer treatment by targeting DNA. However, efficiency is limited due to physico-chemical processes and the insensitivity of native nucleobases to damage. Thus, incorporation of radio- and photosensitizers into these therapies should increase both efficacy and the yield of DNA damage. To date, studies of sensitization processes have been performed on simple model systems, e.g., buffered solutions of dsDNA or sensitizers alone. To fully understand the sensitization processes and to be able to develop new efficient sensitizers in the future, well established model systems are necessary. In the cell environment, DNA tightly interacts with proteins and incorporating this interaction is necessary to fully understand the DNA sensitization process. In this work, we used dsDNA/protein complexes labeled with photo- and radiosensitizers and investigated degradation pathways using LC-MS and HPLC after X-ray or UV radiation.

scholarly journals A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization

2021 ◽  
Author(s):  
Yiran Chang ◽  
Danie J Dickinson
Keyword(s):  
Cell Biology ◽  
Protein Complexes ◽  
Quantitative Model ◽  
Cell Polarization ◽  
Protein Distribution ◽  
Cell Stage ◽  
Viscous Forces ◽  
Dependent Protein ◽  
Regulate Protein ◽  
Size Threshold

Regulation of subcellular components' localization and motion is a critical theme in cell biology. Cells use the actomyosin cortex to regulate protein distribution on the plasma membrane, but the interplay between membrane binding, cortical movements and protein distribution remains poorly understood. In a polarizing one-cell stage Caenorhabditis elegans embryo, actomyosin flows transport PAR protein complexes into an anterior cortical domain to establish the anterior-posterior axis of the animal. Oligomerization of a key scaffold protein, PAR-3, is required for aPAR cortical localization and segregation. Although PAR-3 oligomerization is essential for polarization, it remains unclear how oligomer size contributes to aPAR segregation because PAR-3 oligomers are a heterogeneous population of many different sizes. To address this question, we engineered PAR-3 to defined sizes. We report that PAR-3 trimers are necessary and sufficient for PAR-3 function during polarization and later embryo development, while larger PAR-3 clusters are dispensable. Quantitative analysis of PAR-3 diffusion showed that PAR-3 clusters larger than a trimer are transported by viscous forces without being physically captured by the actomyosin cortex. Our study provides a quantitative model for size-dependent protein transportation of membrane proteins by cortical flow.

scholarly journals Versatility of USP18 in physiology and pathophysiology

2019 ◽  
Author(s):  
Paulina Dziamałek-Macioszczyk ◽  
Joanna Haraźna ◽  
Tomasz Stompór
Keyword(s):  
Bacterial Infections ◽  
Current Knowledge ◽  
Cell Signalling ◽  
Signalling Pathways ◽  
Type I ◽  
Multifunctional Protein ◽  
Enzymatic Function ◽  
Multiple Processes ◽  
Specific Protease ◽  
Interferon Stimulated Gene 15

Ubiquitin-specific peptidase 18 (USP18) is a multifunctional protein and its roles are still being investigated. This enzyme removes ubiquitin-like molecules from their substrates and the only known interferon-stimulated gene 15 (ISG15) specific protease. Apart from its enzymatic function, it also inhibits interferon type I and III signalling pathways. USP18 is known to regulate multiple processes, such as: cell cycle, cell signalling and response to viral and bacterial infections. Moreover, it contributes to the development of several autoimmune diseases and carcinogenesis, and recently was described as a cardiac remodelling inhibitor. This review summarizes the current knowledge on USP18 functions, highlighting its contribution to the development of heart failure, given the fact that this disease’s etiology is now considered to be inflammatory in nature.

scholarly journals Insulin signalling: putting the G- in protein-protein interactions

2004 ◽  
Vol 380 (1) ◽  
pp. e11-e12 ◽  
Author(s):  
Craig C. MALBON
Keyword(s):  
Insulin Receptor ◽  
Cross Talk ◽  
Protein Interactions ◽  
Tyrosine Kinases ◽  
Cell Biology ◽  
Receptor Tyrosine Kinases ◽  
Insulin Signalling ◽  
Cell Signalling ◽  
Protein Protein Interactions ◽  
Proximal Point

Cell signalling via receptor tyrosine kinases, such as the insulin receptor, and via heterotrimeric G-proteins, such as Gαi, Gαs and Gαq family members, constitute two of most avidly studied paradigms in cell biology. That elements of these two populous signalling pathways must cross-talk to achieve proper signalling in the regulation of cell proliferation, differentiation and metabolism has been anticipated, but the evolution of our thinking and the analysis of such cross-talk have lagged behind the ever-expanding troupe of players and the recognition of multivalency as the rule, rather than the exception, in signalling biology. New insights have been provided by Kreuzer et al. in this issue of the Biochemical Journal, in which insulin is shown to provoke recruitment of Gαi-proteins to insulin-receptor-based complexes that can regulate the gain of insulin-receptor-catalysed autophosphorylation, a proximal point in the insulin-sensitive cascade of signalling. Understanding the convergence and cross-talk of signals from the receptor tyrosine kinases and G-protein-coupled receptor pathways in physical, spatial and temporal contexts will remain a major challenge of cell biology.

scholarly journals The Scissors Model of Microcrack Detection in Bone: Work in Progress

2010 ◽  
Vol 1274 ◽  
Author(s):  
David Taylor ◽  
Lauren Mulcahy ◽  
Gerardo Presbitero ◽  
Pietro Tisbo ◽  
Clodagh Dooley ◽  
...  
Keyword(s):  
Cell Biology ◽  
Cell Signalling ◽  
System Modelling ◽  
Ongoing Research ◽  
Cellular Processes ◽  
Disease States ◽  
Bone Damage ◽  
Model Cell ◽  
Drug Treatments ◽  
Microcrack Detection

AbstractWe have proposed a new model for microcrack detection by osteocytes in bone. According to this model, cell signalling is initiated by the cutting of cellular processes which span the crack. We show that shear displacements of the crack faces are needed to rupture these processes, in an action similar to that of a pair of scissors. Current work involves a combination of cell biology experiments, theoretical and experimental fracture mechanics and system modelling using control theory approaches. The approach will be useful for understanding effects of extreme loading, aging, disease states and drug treatments on bone damage and repair; the present paper presents recent results from experiments and simulations as part of current, ongoing research.

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.

Salmonella in Australia: understanding and controlling infection

2017 ◽  
Vol 38 (3) ◽  
pp. 112
Author(s):  
Joshua PM Newson
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Protein Translocation ◽  
Cell Signalling ◽  
Effector Proteins ◽  
Host Cells ◽  
Secretion Systems ◽  
Significant Burden ◽  
Systemic Infections ◽  
Bacterial Effectors

The bacterium Salmonella causes a spectrum of foodborne diseases ranging from acute gastroenteritis to systemic infections, and represents a significant burden of disease globally. In Australia, Salmonella is frequently associated with outbreaks and is a leading cause of foodborne illness, which results in a significant medical and economic burden. Salmonella infection involves colonisation of the small intestine, where the bacteria invades host cells and establishes an intracellular infection. To survive within host cells, Salmonella employs type-three secretion systems to deliver bacterial effector proteins into the cytoplasm of host cells. These bacterial effectors seek out and modify specific host proteins, disrupting host processes such as cell signalling, intracellular trafficking, and programmed cell death. This strategy of impairing host cells allows Salmonella to establish a replicative niche within the cell, where they can replicate to high numbers before escaping to infect neighbouring cells, or be transmitted to new hosts. While the importance of effector protein translocation to infection is well established, our understanding of many effector proteins remains incomplete. Many Salmonella effectors have unknown function and unknown roles during infection. A greater understanding of how Salmonella manipulates host cells during infection will lead to improved strategies to prevent, control, and eliminate disease. Further, studying effector proteins can be a useful means for exploring host cell biology and elucidating the details of host cell signalling.

scholarly journals STRIPAK, a highly conserved signaling complex, controls multiple eukaryotic cellular and developmental processes and is linked with human diseases

2019 ◽  
Vol 400 (8) ◽  
pp. 1005-1022 ◽  
Author(s):  
Ulrich Kück ◽  
Daria Radchenko ◽  
Ines Teichert
Keyword(s):  
Protein Complexes ◽  
Model Systems ◽  
Human Diseases ◽  
Physical Interaction ◽  
Developmental Processes ◽  
Signaling Complex ◽  
Macromolecular Assembly ◽  
Complex Functions ◽  
Key Issues ◽  
Eukaryotic Organisms

Abstract The striatin-interacting phosphatases and kinases (STRIPAK) complex is evolutionary highly conserved and has been structurally and functionally described in diverse lower and higher eukaryotes. In recent years, this complex has been biochemically characterized better and further analyses in different model systems have shown that it is also involved in numerous cellular and developmental processes in eukaryotic organisms. Further recent results have shown that the STRIPAK complex functions as a macromolecular assembly communicating through physical interaction with other conserved signaling protein complexes to constitute larger dynamic protein networks. Here, we will provide a comprehensive and up-to-date overview of the architecture, function and regulation of the STRIPAK complex and discuss key issues and future perspectives, linked with human diseases, which may form the basis of further research endeavors in this area. In particular, the investigation of bi-directional interactions between STRIPAK and other signaling pathways should elucidate upstream regulators and downstream targets as fundamental parts of a complex cellular network.

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