scholarly journals Rational design of minimal synthetic promoters for plants

2020 ◽  
Vol 48 (21) ◽  
pp. 11845-11856 ◽  
Author(s):  
Yao-Min Cai ◽  
Kalyani Kallam ◽  
Henry Tidd ◽  
Giovanni Gendarini ◽  
Amanda Salzman ◽  
...  
Keyword(s):  
Rational Design ◽  
Plant Pathogens ◽  
Critical Role ◽  
Experimental System ◽  
Computational Design ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Synthetic Promoters ◽  
Sequence Elements ◽  
Plant Promoters

Abstract Promoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors. Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilize plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling the computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

scholarly journals Rational Design of Minimal Synthetic Promoters for Plants

2020 ◽  
Author(s):  
Yaomin Cai ◽  
Kalyani Kallam ◽  
Henry Tidd ◽  
Giovanni Gendarini ◽  
Amanda Salzman ◽  
...  
Keyword(s):  
Rational Design ◽  
Plant Pathogens ◽  
Critical Role ◽  
Experimental System ◽  
Computational Design ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Synthetic Promoters ◽  
Sequence Elements ◽  
Plant Promoters

ABSTRACTPromoters serve a critical role in establishing baseline transcriptional capacity through the recruitment of proteins, including transcription factors (TFs). Previously, a paucity of data for cis-regulatory elements in plants meant that it was challenging to determine which sequence elements in plant promoter sequences contributed to transcriptional function. In this study, we have identified functional elements in the promoters of plant genes and plant pathogens that utilise plant transcriptional machinery for gene expression. We have established a quantitative experimental system to investigate transcriptional function, investigating how identity, density and position contribute to regulatory function. We then identified permissive architectures for minimal synthetic plant promoters enabling computational design of a suite of synthetic promoters of different strengths. These have been used to regulate the relative expression of output genes in simple genetic devices.

scholarly journals Rational design and testing of abiotic stress‐inducible synthetic promoters from poplar cis ‐regulatory elements

2021 ◽  
Author(s):  
Yongil Yang ◽  
Jun Hyung Lee ◽  
Magen R. Poindexter ◽  
Yuanhua Shao ◽  
Wusheng Liu ◽  
...  
Keyword(s):  
Abiotic Stress ◽  
Rational Design ◽  
Regulatory Elements ◽  
Synthetic Promoters ◽  
Design And Testing

scholarly journals Improving plant transient expression through the rational design of synthetic 5′ and 3′ untranslated regions

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Hadrien Peyret ◽  
James K. M. Brown ◽  
George P. Lomonossoff
Keyword(s):  
Recombinant Protein ◽  
Transient Expression ◽  
Recombinant Proteins ◽  
Rational Design ◽  
Critical Role ◽  
Regulatory Elements ◽  
Plant Viruses ◽  
Untranslated Regions ◽  
Expression Vectors ◽  
Expression Systems

Abstract Background The growing field of plant molecular farming relies on expression vectors that allow high yields of recombinant proteins to be produced through transient gene expression. While numerous expression vectors currently exist for this purpose, there are very few examples of systematic efforts to improve upon these. Moreover, the current generation of expression systems makes use of naturally-occurring regulatory elements, typically selected from plant viruses, to maximise yields. This study aims to use rational design to generate synthetic sequences that can rival existing ones. Results In this work, we present the rational design of novel synthetic 5′ and 3′ untranslated regions (UTRs) which can be used in various combinations to modulate accumulation levels of transiently-expressed recombinant proteins. Using the pEAQ-HT expression vector as a point of comparison, we show that pre-existing expression systems can be improved by the deployment of rationally designed synthetic UTRs. Notably, we show that a suite of short, synthetic 5′UTRs behave as expression enhancers that outperform the HT 5′UTR present in the CPMV-HT expression system. Furthermore, we confirm the critical role played by the 3′UTR of cowpea mosaic virus RNA-2 in the performance of the CPMV-HT system. Finally, we use the knowledge obtained from these results to develop novel expression vectors (named pHRE and pHREAC) that equal or outperform pEAQ-HT in terms of recombinant protein yield. These new vectors are also domesticated for the use of certain Type IIS restriction enzymes, which allows for quicker cloning and straightforward assessment of different combinations of UTRs. Conclusions We have shown that it is possible to rationally design a suite of expression modulators in the form of synthetic UTRs. We have created novel expression vectors that allow very high levels of recombinant protein expression in a transient expression context. This will have important consequences for future efforts to develop ever-better plant transient overexpression vectors for research or industrial applications.

Molecular Design of Peptide-Fc Fusion Drugs

2019 ◽  
Vol 20 (3) ◽  
pp. 203-208 ◽  
Author(s):  
Lin Ning ◽  
Bifang He ◽  
Peng Zhou ◽  
Ratmir Derda ◽  
Jian Huang
Keyword(s):  
Rational Design ◽  
Molecular Design ◽  
Computational Design ◽  
Fusion Technology ◽  
Computational Tools ◽  
Open Questions ◽  
Biological Therapeutics ◽  
Key Steps ◽  
Computational Resources ◽  
Tools And Methods

Background:Peptide-Fc fusion drugs, also known as peptibodies, are a category of biological therapeutics in which the Fc region of an antibody is genetically fused to a peptide of interest. However, to develop such kind of drugs is laborious and expensive. Rational design is urgently needed.Methods:We summarized the key steps in peptide-Fc fusion technology and stressed the main computational resources, tools, and methods that had been used in the rational design of peptide-Fc fusion drugs. We also raised open questions about the computer-aided molecular design of peptide-Fc.Results:The design of peptibody consists of four steps. First, identify peptide leads from native ligands, biopanning, and computational design or prediction. Second, select the proper Fc region from different classes or subclasses of immunoglobulin. Third, fuse the peptide leads and Fc together properly. At last, evaluate the immunogenicity of the constructs. At each step, there are quite a few useful resources and computational tools.Conclusion:Reviewing the molecular design of peptibody will certainly help make the transition from peptide leads to drugs on the market quicker and cheaper.

scholarly journals Computational-Driven Epitope Verification and Affinity Maturation of TLR4-Targeting Antibodies

2021 ◽  
Vol 22 (11) ◽  
pp. 5989
Author(s):  
Bilal Ahmad ◽  
Maria Batool ◽  
Moon Suk Kim ◽  
Sangdun Choi
Keyword(s):  
Rational Design ◽  
Structural Integrity ◽  
Critical Role ◽  
Autoimmune Encephalitis ◽  
Affinity Maturation ◽  
Antibody Binding ◽  
Computational Techniques ◽  
Multiple Strategies ◽  
Binding Epitope

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody–antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.

scholarly journals Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 911
Author(s):  
Joana Silva ◽  
Pedro Nina ◽  
Luísa Romão
Keyword(s):  
Translational Regulation ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Leader Sequence ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequence ◽  
Abc Protein ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

scholarly journals RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

2009 ◽  
Vol 20 (1) ◽  
pp. 410-418 ◽  
Author(s):  
Ulf R. Klein ◽  
Markus Haindl ◽  
Erich A. Nigg ◽  
Stefan Muller
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Critical Role ◽  
Specific Interaction ◽  
Regulatory Function ◽  
Mitotic Progression ◽  
Chromosome Congression ◽  
Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

scholarly journals The decapentaplegic core promoter region plays an integral role in the spatial control of transcription.

1995 ◽  
Vol 15 (7) ◽  
pp. 3960-3968 ◽  
Author(s):  
D H Schwyter ◽  
J D Huang ◽  
T Dubnicoff ◽  
A J Courey
Keyword(s):  
Expression Pattern ◽  
Phase Ii ◽  
Promoter Region ◽  
Core Promoter ◽  
Critical Role ◽  
Regulatory Elements ◽  
Drosophila Embryo ◽  
Phase Iii ◽  
Core Promoter Region ◽  
Flanking Region

The Drosophila melanogaster decapentaplegic (dpp) gene encodes a transforming growth factor beta-related cell signaling molecule that plays a critical role in dorsal/ventral pattern formation. The dpp expression pattern in the Drosophila embryo is dynamic, consisting of three phases. Phase I, in which dpp is expressed in a broad dorsal domain, depends on elements in the dpp second intron that interact with the Dorsal transcription factor to repress transcription ventrally. In contrast, phases II and III, in which dpp is expressed first in broad longitudinal stripes (phase II) and subsequently in narrow longitudinal stripes (phase III), depend on multiple independent elements in the dpp 5'-flanking region. Several aspects of the normal dpp expression pattern appear to depend on the unique properties of the dpp core promoter. For example, this core promoter (extending from -22 to +6) is able to direct a phase II expression pattern in the absence of additional upstream or downstream regulatory elements. In addition, a ventral-specific enhancer in the dpp 5'-flanking region that binds the Dorsal factor activates the heterologous hsp70 core promoter but not the dpp core promoter. Thus, the dpp core promoter region may contribute to spatially regulated transcription both by interacting directly with spatially restricted activators and by modifying the activity of proteins bound to enhancer elements.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences

1988 ◽  
Vol 8 (9) ◽  
pp. 3827-3836
Author(s):  
N P Williams ◽  
P P Mueller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Yeast Genes ◽  
Reading Frames

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1189-1197 ◽  
Author(s):  
Hua Tang ◽  
Zhenhong Guo ◽  
Minghui Zhang ◽  
Jianli Wang ◽  
Guoyou Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Regulatory Function ◽  
Hematopoietic Stem ◽  
Regulatory Dendritic Cells

Abstract Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11bhiIalo DCs secreting high levels of TGF-β, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11bhiIalo DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11bhiIalo DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11bhiIalo DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.

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