scholarly journals Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease

2020 ◽  
Vol 117 (52) ◽  
pp. 33186-33196
Author(s):  
Mateo I. Sanchez ◽  
Quynh-Anh Nguyen ◽  
Wenjing Wang ◽  
Ivan Soltesz ◽  
Alice Y. Ting
Keyword(s):  
First Generation ◽  
Dynamic Range ◽  
Single Chain ◽  
Tev Protease ◽  
Cellular Manipulation ◽  
Single Polypeptide Chain ◽  
Cellular Events ◽  
Protease Cleavage ◽  
Single Polypeptide

Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain–caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.

scholarly journals Structural diversity of oligomeric β-propellers with different numbers of identical blades

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Evgenia Afanasieva ◽  
Indronil Chaudhuri ◽  
Jörg Martin ◽  
Eva Hertle ◽  
Astrid Ursinus ◽  
...  
Keyword(s):  
High Resolution ◽  
Building Block ◽  
Polypeptide Chain ◽  
Structural Diversity ◽  
Building Blocks ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Data Support ◽  
Single Polypeptide ◽  
Structural Versatility

β-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One- to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High-resolution structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, respectively, show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.

scholarly journals Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies

1984 ◽  
Vol 218 (2) ◽  
pp. 601-608 ◽  
Author(s):  
T Lah ◽  
M Drobniĉ-Koŝorok ◽  
V Turk ◽  
R H Pain
Keyword(s):  
Cathepsin D ◽  
Specific Activity ◽  
Single Chain ◽  
Guanidinium Chloride ◽  
Single Polypeptide Chain ◽  
Acid Environment ◽  
Single Polypeptide ◽  
Kinetics Of ◽  
Chain Form ◽  
Covalent Complex

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

scholarly journals Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single chain diabody

2021 ◽  
Author(s):  
Jessica Krakow ◽  
Michal Hammel ◽  
Ying Zhu ◽  
Brian J Hillier ◽  
Bryce Paolella ◽  
...  
Keyword(s):  
T Cell ◽  
Single Chain ◽  
Cleavage Rate ◽  
Post Translational Modifications ◽  
Single Polypeptide Chain ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Single Domain Antibodies ◽  
Single Polypeptide ◽  
Structural Architecture

Abstract Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

scholarly journals Contextual reprogramming of CAR-T cells for treatment of HER2+ cancers

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Zhifen Yang ◽  
Lingyu Li ◽  
Ahu Turkoz ◽  
Pohan Chen ◽  
Rona Harari-Steinfeld ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor Receptor ◽  
Checkpoint Inhibition ◽  
Single Chain ◽  
Tev Protease ◽  
Car T Cells ◽  
Car T

Abstract Background Adoptive transfer of chimeric antigen receptor (CAR)-engineered T cells combined with checkpoint inhibition may prevent T cell exhaustion and improve clinical outcomes. However, the approach is limited by cumulative costs and toxicities. Methods To overcome this drawback, we created a CAR-T (RB-340-1) that unites in one product the two modalities: a CRISPR interference-(CRISPRi) circuit prevents programmed cell death protein 1 (PD-1) expression upon antigen-encounter. RB-340-1 is engineered to express an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3ζ co-stimulatory domains linked to the tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the PD-1 transcription start site (TSS). A second constructs includes linker for activation of T cells (LAT) fused to nuclease-deactivated spCas9 (dCas9)-Kruppel-associated box (KRAB) via a TEV-cleavable sequence (TCS). Upon antigen encounter, the LAT-dCas9-KRAB (LdCK) complex is cleaved by TEV allowing targeting of dCas9-KRAB to the PD-1 gene TSS. Results Here, we show that RB-340-1 consistently demonstrated higher production of homeostatic cytokines, enhanced expansion of CAR-T cells in vitro, prolonged in vivo persistence and more efficient suppression of HER2+ FaDu oropharyngeal cancer growth compared to the respective conventional CAR-T cell product. Conclusions As the first application of CRISPRi toward a clinically relevant product, RB-340-1 with the conditional, non-gene editing and reversible suppression promotes CAR-T cells resilience to checkpoint inhibition, and their persistence and effectiveness against HER2-expressing cancer xenografts.

SINGLE-CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR (SCU-PA) FROM HT-1080 HUMAN FIBROSARCOMA CELLS IS A GENUINE PROENZYME

1987 ◽  
Author(s):  
L Skriver ◽  
L C Petersen ◽  
L R Lund ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pancreatic Trypsin Inhibitor ◽  
Human Fibrosarcoma Cells ◽  
Single Polypeptide ◽  
Hydrolysis Of

U-PA is released from many cells as a single polypeptide chain (scu-PA) that is converted into its active two-chain form (tcu-PA) by limited proteolysis with plasmin. There is general agreement that scu-PA has an extremely low amidolytic activity, but different oppinions exist, as to whether scu-PA itself can activate plasminogen. We have reinvestigated the plasminogen activating activity of our scu-PA preparations by means of a direct [125]I-plasminogen conversion assay and two amidolytic assays for plasmin and u-PA activity. In the [125]I-plasminogen conversion assay in the presence of bovine pancreatic trypsin inhibitor (BPTI) the subsequent plasmin catalyzed conversion of scu-PA is blocked while the plasminogen activation is unaffected. In this assay with 3oo nM Glu-plasminogen and 15 pM BPTI, 4o nM scu-PA caused a low but significant plasminogen conversion, which could be fully inhibited by pretreatment of scu-PA with diisopropylfluorophos-phate (DFP). DFP-treated scu-PA was convertible to fully active tcu-PA. Rates of plasminogen activation in this type of assay for scu-PA activity was at least 4oo fold slower than that measured for tcu-PA. A coupled amidolytic assay with Lys-plasminogen, scuPA or tcu-PA, BPTI, and the high affinity plasmin substrate H-D-Val-Phe-LyspNA (S2390) was performed under conditions that ensures a low steady state concentration of free plasmin. In this assay the initial rate of Lys-plasminogen activation by DFP-treated scu-PA was at least 25o fold slower than that measured for tcu-PA. Finally, u-PA activity was measured in an assay with the chromogenic substrate <Glu-Gly-ArgpNA (S2444) (o.8mM) in the presence of highly purified Glu-plasminogen (3oonM) and DFP-treated scu-PA (2nM) in the absence of BPTI. Within the initial 15 min of incubation no detectable hydrolysis of S2444 occurred. Addition of tcu-PA (2pM) or plasmin (o.lnM) to the scu-PA/Glu-plasminogen mixture caused a significant reduction of the lag period before onset of the cascade reaction leading to scu-PA conversion and subsequent hydrolysis of S2444. We conclude that the low rates of plasminogen activation measured in these assays by scu-PA might be accounted for by the presence of trace amounts of tcu-PA in the scu-PA preparations, and that scu-PA meets the requirements for a genuine proenzyme

scholarly journals Design: An assay based on single-polypeptide-chain heterodimeric A2AR/D2R and non-oligomerized fusions for in vivo analysis of their allosteric receptor-receptor interactions

2016 ◽  
Author(s):  
Toshio Kamiya ◽  
Takashi Masuko ◽  
Dasiel Oscar Borroto-Escuela ◽  
Haruo Okado ◽  
Hiroyasu Nakata
Keyword(s):  
Energy Transfer ◽  
Polypeptide Chain ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
R And D ◽  
Antenna Complex ◽  
Single Polypeptide Chain ◽  
Single Polypeptide ◽  
Light Harvesting Antenna

AbstractBackgroundThe adenosine A2A receptor (A2AR) heteromerizes with the dopamine D2 receptor (D2R). In order to explore their functional interaction, we engineered previously stable single-polypeptide-chain (sc) A2AR/D2LR: whether the molecular entity of the striatal A2AR/D2R antagonism, i.e., scA2AR/D2Rs are just A2AR/D2R with the antagonism, remains unresolved.New MethodTo further clarify the heteromerization through the scA2AR/D2LR, we here designed supramolecularly ‘exclusive’ monomers and dimers, using the Cε2 domain of IgE-Fc or apoproteins of the bacterial light-harvesting antenna complex.ResultsA concept of the recptor protein assembly regulation, i.e., the selective monomer/non-obligate dimer formation was obtained. Although none of these new fusions were constructed or tested, we could aim at obtaining heterodimer-specific agents, using the scA2AR/D2R. Whether the resulting designs were explained feasibly and rationally was addressed. The structure and function of the non-obligate dimer were here discussed through scA2AR/D2R, focusing on the procedure of the membrane protein design and methods for transient protein-protein interactions.Summary and OutlookGiven that upon being expressed and allosteric regulation occurs regardless of specific signal to non-specific noise (S/N) ratio, the supramolecular designs, allowing us to express selectively monomer/non-obligate dimer of class A GPCR, are experimentally testable and will be used to confirm in vivo that such low S/N ratio interaction between A2AR and D2LR functions in the dopamine neurotransmission in the striatum.Abbreviations:A2ARadenosine A2A receptor3HA-A2ARA2AR tagged with a triple HA epitopeBchlbacteriochlorophyllBRETbioluminescence resonance energy transferCcarboxy-terminalCDcluster of differentiationD2LR and D2SRthe long and short form of dopamine D2 receptor, respectivelyFabantigen binding fragmentFcFc fragmentFcεRIhigh affinity receptor for IgEFRETfluorescence resonance energy transferG4San amino acid sequence consisting of a four-glycine-repeat followed by a serine residueGABAγ-aminobutyric acidGABABGABA type B receptorGPCRG protein-coupled receptorGttransducinHAhemagglutininHIVhuman immunodeficiency virusICintracellular loopsIgimmunoglobulinLHlight-harvesting antenna complexmAbmonoclonal antibodyMrmolecular weightNamino-terminalPDParkinson’s diseasePSphotosystemRCreaction centerRlucRenilla luciferasescsingle-chainTMtransmembrane3Dthree-dimensional

scholarly journals Structural diversity of oligomeric β-propellers with different numbers of identical blades

2019 ◽  
Author(s):  
Evgenia Afanasieva ◽  
Indronil Chaudhuri ◽  
Jörg Martin ◽  
Eva Hertle ◽  
Astrid Ursinus ◽  
...  
Keyword(s):  
High Resolution ◽  
Building Block ◽  
Polypeptide Chain ◽  
Structural Diversity ◽  
Building Blocks ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Data Support ◽  
Single Polypeptide ◽  
Structural Versatility

Abstractβ-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One-to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High-resolution structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, respectively, show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.

scholarly journals Tetravalent single-chain avidin: from subunits to protein domains via circularly permuted avidins

2005 ◽  
Vol 392 (3) ◽  
pp. 485-491 ◽  
Author(s):  
Henri R. Nordlund ◽  
Vesa P. Hytönen ◽  
Jarno Hörhä ◽  
Juha A. E. Määttä ◽  
Daniel J. White ◽  
...  
Keyword(s):  
Binding Sites ◽  
Polypeptide Chain ◽  
Protein Complexes ◽  
Fusion Partner ◽  
Wild Type ◽  
Single Chain ◽  
Binding Domains ◽  
Single Polypeptide Chain ◽  
Biotin Binding ◽  
Single Polypeptide

scAvd (single-chain avidin, where two dcAvd are joined in a single polypeptide chain), having four biotin-binding domains, was constructed by fusion of topologically modified avidin units. scAvd showed similar biotin binding and thermal stability properties as chicken avidin. The DNA construct encoding scAvd contains four circularly permuted avidin domains, plus short linkers connecting the four domains into a single polypeptide chain. In contrast with wild-type avidin, which contains four identical avidin monomers, scAvd enables each one of the four avidin domains to be independently modified by protein engineering. Therefore the scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudotetrameric avidin molecules showing different domain characteristics. In addition, unmodified scAvd could be used as a fusion partner, since it provides a unique non-oligomeric structure, which is fully functional with four high-affinity biotin-binding sites. Furthermore, the subunit-to-domain strategy described in the present study could be applied to other proteins and protein complexes, facilitating the development of sophisticated protein tools for applications in nanotechnology and life sciences.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

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