In Silico Evaluation of Cyclophilin Inhibitors as Potential Treatment for SARS-CoV-2

Abstract The advent of SARS-CoV-2 provoked researchers to propose multiple antiviral strategies to improve patients' outcomes. Studies provide evidence that Cyclosporine A (CsA) decreases SARS-CoV-2 replication in vitro, and decreases mortality rates of COVID-19 patients. CsA binds Cyclophilins, which isomerize prolines, affecting viral protein activity. We investigated the proline composition from various Coronavirus proteomes to identify proteins that may critically rely on cyclophilin’s peptidyl-proline isomerase activity and found the Nucleocapsid (N) protein significantly depends on Cyclophilin A (CyPA). We modeled CyPA and N protein interactions to demonstrate the N protein as a potential indirect therapeutic target of CsA, which we propose may impede coronavirus replication by obstructing nucleocapsid folding. Finally, we analyzed literature and protein-protein interactions, finding evidence that, by inhibiting CyPA, CsA may impact coagulation proteins and hemostasis. Despite CsA’s promising antiviral characteristics, the interactions between cyclophilins and coagulation factors emphasize risk stratification for COVID patients with thrombosis dispositions.

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Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA.

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4168 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4168-4178 ◽

Cited By ~ 22

Author(s):

C L Peterson ◽

K Orth ◽

K L Calame

Keyword(s):

Heavy Chain ◽

Protein Interactions ◽

Binding Sites ◽

Immunoglobulin Heavy Chain ◽

Lymphoid Cells ◽

Protein Protein Interactions ◽

Protein Activity ◽

Enhancer Element ◽

Enhancer Sequences

Seven protein-binding sites on the immunoglobulin heavy-chain (IgH) enhancer element have been identified by exonuclease III protection and gel retardation assays. It appears that the seven sites bind a minimum of four separate proteins. Three of these proteins also bind to other enhancers or promoters, but one protein seems to recognize exclusively IgH enhancer sequences. A complex of four binding sites, recognized by different proteins, is located within one 80-base-pair region of IgH enhancer DNA. Close juxtaposition of enhancer proteins may allow protein-protein interactions or be part of a mechanism for modulating enhancer protein activity. All IgH enhancer-binding proteins identified in this study were found in extracts from nonlymphoid as well as lymphoid cells. These data provide the first direct evidence that multiple proteins bind to enhancer elements and that while some of these proteins recognize common elements of many enhancers, others have more limited specificities.

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In silico study of SARS-CoV-2 Nucleocapsid Protein-Protein Interactions and Potential Candidates for their Stabilization

10.20944/preprints202007.0558.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Martin Lavecchia ◽

Julian Fernandez

Keyword(s):

Protein Interactions ◽

Nucleocapsid Protein ◽

Drug Repurposing ◽

Structural Motif ◽

Protein Protein Interactions ◽

Health Crisis ◽

N Protein ◽

Worldwide Distribution ◽

Novel Coronavirus

The outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19, has caused a global health crisis. Unfortunately, only a few treatments have proved to be effective, and their worldwide distribution remains as a challenge. Due to the urgency of the situation, drug repurposing remains as the fastest way to identify possible therapeutic options. Recent studies have shown that the stabilization of non-native Protein-Protein Interactions (PPIs) of the nucleocapsid protein of MERS coronavirus is a valid strategy to inhibit viral replication, but no study up to date has been done in SARS-CoV-2. In this work, a novel protocol for the discovery of PPIs stabilizers is presented and applied to SARS-CoV-2 N protein with a drug repurposing approach. This enabled us to identify that catechin, a structural motif present in widely distributed natural products, might be a privileged scaffold for this type of stabilization. Since many of the compounds presented in this work are generally considered nutraceuticals and have also been exhaustively studied, even though some of them contain PAINS substructures, could be good candidates for the SARS-CoV-2 nucleocapsid inhibition and be considered for further in vitro testing against COVID-19.

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Binding in vitro of multiple cellular proteins to immunoglobulin heavy-chain enhancer DNA

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4168-4178.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4168-4178

Author(s):

C L Peterson ◽

K Orth ◽

K L Calame

Keyword(s):

Heavy Chain ◽

Protein Interactions ◽

Binding Sites ◽

Immunoglobulin Heavy Chain ◽

Lymphoid Cells ◽

Protein Protein Interactions ◽

Protein Activity ◽

Enhancer Element ◽

Enhancer Sequences

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target

Biomolecules ◽

10.3390/biom11040510 ◽

2021 ◽

Vol 11 (4) ◽

pp. 510

Author(s):

Maho Yamamoto ◽

Rina Kondo ◽

Haruka Hozumi ◽

Seita Doi ◽

Miwako Denda ◽

...

Keyword(s):

Protein Interactions ◽

Biochemical Characterization ◽

S100 Protein ◽

High Mobility ◽

Dependent Manner ◽

Protein Signaling ◽

Protein Protein Interactions ◽

High Mobility Group Protein ◽

Pulldown Assay

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

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Ways into Understanding HIF Inhibition

Cancers ◽

10.3390/cancers13010159 ◽

2021 ◽

Vol 13 (1) ◽

pp. 159

Author(s):

Tina Schönberger ◽

Joachim Fandrey ◽

Katrin Prost-Fingerle

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Protein Protein Interactions ◽

Low Oxygen ◽

Drug Candidates ◽

Open Questions ◽

Wide Range ◽

Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

British Journal of Cancer ◽

10.1038/s41416-020-01250-4 ◽

2021 ◽

Author(s):

Liqing Jia ◽

Xiaolu Ge ◽

Chao Du ◽

Linna Chen ◽

Yanhong Zhou ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Protein Interactions ◽

Elongation Factor ◽

Protein Translation ◽

Protein Protein Interactions ◽

Promising Target ◽

Tgfβ Receptor ◽

Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6021-6029.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6021-6029

Author(s):

R Metz ◽

A J Bannister ◽

J A Sutherland ◽

C Hagemeier ◽

E C O'Rourke ◽

...

Keyword(s):

Protein Interactions ◽

Transcriptional Activation ◽

Binding Protein ◽

Tata Box ◽

Binding Motif ◽

Protein Protein Interactions ◽

High Concentrations ◽

Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

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