scholarly journals Identification and characterization of novel filament-forming proteins in cyanobacteria

2019 ◽  
Author(s):  
Benjamin L. Springstein ◽  
Christian Woehle ◽  
Julia Weissenbach ◽  
Andreas O. Helbig ◽  
Tal Dagan ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Cytoskeletal Proteins ◽  
Tetratricopeptide Repeat ◽  
Cellular Processes ◽  
Tetratricopeptide Repeat Protein ◽  
Pcc 7120 ◽  
Pcc 7942

AbstractFilament-forming proteins in bacteria function in stabilization and localization of proteinaceous complexes and replicons; hence they are instrumental for myriad cellular processes such as cell division and growth. Here we present two novel filament-forming proteins in cyanobacteria. Surveying cyanobacterial genomes for coiled-coil-rich proteins (CCRPs) that are predicted as putative filament-forming proteins, we observed a higher proportion of CCRPs in filamentous cyanobacteria in comparison to unicellular cyanobacteria. Using our predictions, we identified nine protein families with putative intermediate filament (IF) properties. Polymerization assays revealed four proteins that formed polymers in vitro and three proteins that formed polymers in vivo. Fm7001 from Fischerella muscicola PCC 7414 polymerized in vitro and formed filaments in vivo in several organisms. Additionally, we identified a tetratricopeptide repeat protein - All4981 - in Anabaena sp. PCC 7120 that polymerized into filaments in vitro and in vivo. All4981 interacts with known cytoskeletal proteins and is indispensable for Anabaena viability. Although it did not form filaments in vitro, Syc2039 from Synechococcus elongatus PCC 7942 assembled into filaments in vivo and a Δsyc2039 mutant was characterized by an impaired cytokinesis. Our results expand the repertoire of known prokaryotic filament-forming CCRPs and demonstrate that cyanobacterial CCRPs are involved in cell morphology, motility, cytokinesis and colony integrity.

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.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

In vitro and in vivo characterization of graphene oxide coated porcine bone granules

Carbon ◽  
2016 ◽  
Vol 103 ◽  
pp. 291-298 ◽  
Author(s):  
Valeria Ettorre ◽  
Patrizia De Marco ◽  
Susi Zara ◽  
Vittoria Perrotti ◽  
Antonio Scarano ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
In Vivo Characterization

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Preclinical characterization of dostarlimab, a therapeutic anti-PD-1 antibody with potent activity to enhance immune function in in vitro cellular assays and in vivo animal models

mAbs ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 1954136
Author(s):  
Sujatha Kumar ◽  
Srimoyee Ghosh ◽  
Geeta Sharma ◽  
Zebin Wang ◽  
Marilyn R. Kehry ◽  
...  
Keyword(s):  
Animal Models ◽  
Immune Function ◽  
Cellular Assays ◽  
In Vivo Animal Models

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.

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