Rewiring cell signalling through chimaeric regulatory protein engineering

Bacterial cells continuously sense and respond to their environment using their inherent signalling and gene regulatory networks. Cells are equipped with parallel signalling pathways, which can specifically cope with individual input signals, while interconnectivities between pathways lead to an enhanced complexity of regulatory responses that enable sophisticated adaptation. In principle, any cell signalling pathway may be rewired to respond to non-cognate signals by exchanging and recombining their underlying cognate signalling components. In the present article, we review the engineering strategies and use of chimaeric regulatory proteins in cell signalling pathways, especially the TCS (two-component signalling) system in bacteria, to achieve novel customized signalling or regulatory functions. We envisage that engineered chimaeric regulatory proteins can play an important role to aid both forward and reverse engineering of biological systems for many desired applications.

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Mutations in Transcriptional Regulators Allow Selective Engineering of Signal Integration Logic

mBio ◽

10.1128/mbio.01171-14 ◽

2014 ◽

Vol 5 (3) ◽

Cited By ~ 4

Author(s):

Szabolcs Semsey

Keyword(s):

Gene Regulatory Networks ◽

Regulatory Networks ◽

Regulatory Protein ◽

Point Mutations ◽

Building Blocks ◽

Regulatory Elements ◽

Regulatory Proteins ◽

Single Amino Acid ◽

Signal Integration ◽

Gene Regulatory

ABSTRACT Bacterial cells monitor their environment by sensing a set of signals. Typically, these environmental signals affect promoter activities by altering the activity of transcription regulatory proteins. Promoters are often regulated by more than one regulatory protein, and in these cases the relevant signals are integrated by certain logic. In this work, we study how single amino acid substitutions in a regulatory protein (GalR) affect transcriptional regulation and signal integration logic at a set of engineered promoters. Our results suggest that point mutations in regulatory genes allow independent evolution of regulatory logic at different promoters. IMPORTANCE Gene regulatory networks are built from simple building blocks, such as promoters, transcription regulatory proteins, and their binding sites on DNA. Many promoters are regulated by more than one regulatory input. In these cases, the inputs are integrated and allow transcription only in certain combinations of input signals. Gene regulatory networks can be easily rewired, because the function of cis-regulatory elements and promoters can be altered by point mutations. In this work, we tested how point mutations in transcription regulatory proteins can affect signal integration logic. We found that such mutations allow context-dependent engineering of signal integration logic at promoters, further contributing to the plasticity of gene regulatory networks.

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The mPTP and its regulatory proteins: final common targets of signalling pathways for protection against necrosis

Cardiovascular Research ◽

10.1093/cvr/cvr302 ◽

2011 ◽

Vol 94 (2) ◽

pp. 181-189 ◽

Cited By ~ 66

Author(s):

T. Miura ◽

M. Tanno

Keyword(s):

Regulatory Proteins ◽

Signalling Pathways

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Targeting cell signalling pathways to fight the flu: towards a paradigm change in anti-influenza therapy

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkp161 ◽

2009 ◽

Vol 64 (1) ◽

pp. 1-4 ◽

Cited By ~ 79

Author(s):

S. Ludwig

Keyword(s):

Cell Signalling ◽

Signalling Pathways ◽

Paradigm Change

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Binding of aldolase to actin-containing filaments. Evidence of interaction with the regulatory proteins of skeletal muscle

Biochemical Journal ◽

10.1042/bj1860089 ◽

1980 ◽

Vol 186 (1) ◽

pp. 89-98 ◽

Cited By ~ 51

Author(s):

T P Walsh ◽

D J Winzor ◽

F M Clarke ◽

C J Masters ◽

D J Morton

Keyword(s):

Skeletal Muscle ◽

Binding Constant ◽

Moving Boundary ◽

Regulatory Protein ◽

Regulatory Proteins ◽

Affinity Constant ◽

Quantitative Studies ◽

Multiple Binding Sites ◽

Multiple Binding ◽

Troponin Complex

The interactions of aldolase with regulatory proteins of rabbit skeletal muscle were investigated by moving-boundary electrophoresis. A salt-dependent interaction of troponin, tropomyosin and the tropomyosin-troponin complex with aldolase was detected, the tropomyosin-troponin complex displaying a greater affinity for the enzyme than did either regulatory protein alone. The results indicate that aldolase possesses multiple binding sites (three or more) for these muscle proteins. Quantitative studies of the binding of aldolase to actin-containing filaments showed the interaction to be influenced markedly by the presence of these muscle regulatory proteins on the filaments. In imidazole/HCl buffer, I 0.088, pH 6.8, aldolase binds to F-actin with an affinity constant of 2 × 10(5) M-1 and a stoicheiometry of one tetrameric aldolase molecule per 14 monomeric actin units. Use of F-actin-tropomyosin as adsorbent results in a doubling of the stoicheiometry without significant change in the intrinsic association constant. With F-actin-tropomyosin-troponin a lower binding constant (6 × 10(4) M-1) but even greater stoicheiometry (4:14 actin units) are observed. The presence of Ca2+ (0.1 mM) decreases this stoicheiometry to 3:14 without affecting significantly the magnitude of the intrinsic binding constant.

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Valproic Acid Inhibits Proliferation and Reduces Invasiveness in Glioma Stem Cells Through Wnt/β Catenin Signalling Activation

Genes ◽

10.3390/genes9110522 ◽

2018 ◽

Vol 9 (11) ◽

pp. 522 ◽

Cited By ~ 9

Author(s):

Gabriele Riva ◽

Chiara Cilibrasi ◽

Riccardo Bazzoni ◽

Massimiliano Cadamuro ◽

Caterina Negroni ◽

...

Keyword(s):

Stem Cells ◽

Valproic Acid ◽

Histone Deacetylase Inhibitors ◽

Cell Signalling ◽

Luciferase Reporter ◽

Signalling Pathways ◽

Boyden Chamber ◽

Glioma Stem Cells ◽

Bioinformatics Analyses ◽

E Cadherin

Glioblastoma is the most common malignant brain tumour in adults. The failure of current therapies can be ascribed to glioma stem cells (GSCs), which can rapidly repopulate the tumour following the initial treatment. The study of histone deacetylase inhibitors, such as valproic acid (VPA), is becoming an attractive field in cancer research. However, the exact mechanisms underlying its anti-cancer effect remain to be elucidated due to its pleiotropic effects on several cell-signalling pathways. Ingenuity Pathway Analysis (IPA) bioinformatics analysis was performed on genome-wide data regarding GSCs methylome to identify the signalling pathways mainly affected by methylation changes induced by VPA. Real time PCR and luciferase reporter assay were used to better investigate VPA effects on Wnt/β-catenin signalling pathway. VPA effect on GSC proliferation was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Trypan blue assays. Finally, VPA impact on GSC motility was demonstrated by Boyden chamber assay and further confirmed evaluating the expression levels or localisation, through western blot or immunofluorescence, of Twist1, Snail1, E-Cadherin and N-Cadherin. The bioinformatics analyses performed on GSCs methylome highlighted that Wnt/β-catenin signalling was affected by the methylation changes induced by VPA, which could influence its activation status. In particular, we pointed out a general activation of this pathway after VPA exposure, which was accompanied by an inhibitory potential on GSCs proliferation. Finally, we also proved VPA’s ability to inhibit GSCs invasion through Snail1 and Twist1 downregulation and E-Cadherin relocalisation. VPA treatment may represent a new, interesting therapeutic approach to affect GSC proliferation and motility, but further investigations are certainly needed.

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Versatility of USP18 in physiology and pathophysiology

Acta Biochimica Polonica ◽

10.18388/abp.2019_2844 ◽

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.

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Subversion of host cell signalling by the protozoan parasiteLeishmania

Parasitology ◽

10.1017/s0031182005008139 ◽

2005 ◽

Vol 130 (S1) ◽

pp. S27-S35 ◽

Cited By ~ 61

Author(s):

D. J. GREGORY ◽

M. OLIVIER

Keyword(s):

Nitric Oxide ◽

Reactive Oxygen Species ◽

Cell Signalling ◽

Protein Tyrosine Phosphatases ◽

Innate Immune ◽

Signalling Pathways ◽

Oxygen Species ◽

Obligate Intracellular ◽

Intracellular Parasites ◽

Intracellular Ca

The protozoaLeishmaniaspp. are obligate intracellular parasites that inhabit the macrophages of their host. Since macrophages are specialized for the identification and destruction of invading pathogens, both directly and by triggering an innate immune response,Leishmaniahave evolved a number of mechanisms for suppressing some critical macrophage activities. In this review, we discuss how various species ofLeishmaniadistort the host macrophage's own signalling pathways to repress the expression of various cytokines and microbicidal molecules (nitric oxide and reactive oxygen species), and antigen presentation. In particular, we describe how MAP Kinase and JAK/STAT cascades are repressed, and intracellular Ca2+and the activities of protein tyrosine phosphatases, in particular SHP-1, are elevated.

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Proteomic Insights into Starvation of Nitrogen-Replete Cells of Nostoc sp. PCC 7120 under β-N-Methylamino-L-Alanine (BMAA) Treatment

Toxins ◽

10.3390/toxins12060372 ◽

2020 ◽

Vol 12 (6) ◽

pp. 372

Author(s):

Olga A. Koksharova ◽

Ivan O. Butenko ◽

Olga V. Pobeguts ◽

Nina A. Safronova ◽

Vadim M. Govorun

Keyword(s):

Molecular Mechanisms ◽

Nitrogen Starvation ◽

Regulatory Protein ◽

Nitrogen Deficiency ◽

Regulatory Proteins ◽

Pii Protein ◽

Significant Difference ◽

Pcc 7120 ◽

Nitrogen Excess ◽

Starvation Conditions

All cyanobacteria produce a neurotoxic non-protein amino acid β-N-methylamino-L-alanine (BMAA). However, the biological function of BMAA in the regulation of cyanobacteria metabolism still remains undetermined. It is known that BMAA suppresses the formation of heterocysts in diazotrophic cyanobacteria under nitrogen starvation conditions, and BMAA induces the formation of heterocyst-like cells under nitrogen excess conditions, by causing the expression of heterocyst-specific genes that are usually “silent” under nitrogen-replete conditions, as if these bacteria receive a nitrogen deficiency intracellular molecular signal. In order to find out the molecular mechanisms underlying this unexpected BMAA effect, we studied the proteome of cyanobacterium Nostoc sp. PCC 7120 grown under BMAA treatment in nitrogen-replete medium. Experiments were performed in two experimental settings: (1) in control samples consisted of cells grown without the BMAA treatment and (2) the treated samples consisted of cells grown with addition of an aqueous solution of BMAA (20 µM). In total, 1567 different proteins of Nostoc sp. PCC 7120 were identified by LC-MS/MS spectrometry. Among them, 80 proteins belonging to different functional categories were chosen for further functional analysis and interpretation of obtained proteomic data. Here, we provide the evidence that a pleiotropic regulatory effect of BMAA on the proteome of cyanobacterium was largely different under conditions of nitrogen-excess compared to its effect under nitrogen starvation conditions (that was studied in our previous work). The most significant difference in proteome expression between the BMAA-treated and untreated samples under different growth conditions was detected in key regulatory protein PII (GlnB). BMAA downregulates protein PII in nitrogen-starved cells and upregulates this protein in nitrogen-replete conditions. PII protein is a key signal transduction protein and the change in its regulation leads to the change of many other regulatory proteins, including different transcriptional factors, enzymes and transporters. Complex changes in key metabolic and regulatory proteins (RbcL, RbcS, Rca, CmpA, GltS, NodM, thioredoxin 1, RpbD, ClpP, MinD, RecA, etc.), detected in this experimental study, could be a reason for the appearance of the “starvation” state in nitrogen-replete conditions in the presence of BMAA. In addition, 15 proteins identified in this study are encoded by genes, which are under the control of NtcA—a global transcriptional regulator—one of the main protein partners and transcriptional regulators of PII protein. Thereby, this proteomic study gives a possible explanation of cyanobacterium starvation under nitrogen-replete conditions and BMAA treatment. It allows to take a closer look at the regulation of cyanobacteria metabolism affected by this cyanotoxin.

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