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17 pages, 1230 KiB

Open AccessArticle

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions

by Paloma Salinas, Sirine Bibak, Raquel Cantos, Lorena Tremiño, Carmen Jerez, Trinidad Mata-Balaguer and Asunción Contreras

Int. J. Mol. Sci. 2024, 25(10), 5429; https://doi.org/10.3390/ijms25105429 - 16 May 2024

Viewed by 1117

Abstract

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric [...] Read more.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a “false positive”, the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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Figure 1
Location of residues (blue spheres) mutated on the PipX structure (chain E of the PDB file 2XG8), which is shown in cartoon representation with the N-terminal TLD/KOW domain and the C-terminal helices coloured in orange and pink, respectively. Full article ">Figure 2
Effect of PipX point mutations (*) on two-hybrid interaction signals with PII and NtcA. The colour scale, from no interaction (−) to the highest (+) interaction signals, is shown on the left. In each case, representative photographs from a minimum of six assays and heatmaps summarizing the BACTH results on MacConkey-lactose or M63-maltose-X-gal are shown from left to right. The relative position of the T18 or T25 domains is illustrated in each case. The Y2H column indicates the impact of the corresponding mutations in reported Y2H assays: no effect, significant effect and very drastic effect are indicated by an equals sign, one, or two arrows, respectively. Full article ">Figure 3
Location on the PipX surface of mutated residues for which the BACTH system was particularly informative. The surface structure of the PipX subunit (chain E of the PDB file 2XG8) is represented in semi-transparent form, rendering visible the flexed C-terminal helices in cartoon representation. Surface regions of PipX corresponding to mutated residues with discordant results are coloured in red. Full article ">Figure 4
PipX interacts with GlnB and GlnK proteins in the BACTH system. Heatmaps summarise the results for the indicated fusion proteins. Additional data are provided in <a href="#app1-ijms-25-05429" class="html-app">Figure S1 in the Supplementary Materials</a>. Other details are as shown in <a href="#ijms-25-05429-f002" class="html-fig">Figure 2</a>. Full article ">Figure 5
PII gives weaker self- and cross-interaction signals than GlnB or GlnK in the BACTH system. T25 derivatives were expressed from pKT25 vector (A) or pT25 vector (B). Additional data are provided in <a href="#app1-ijms-25-05429" class="html-app">Figure S1 in the Supplementary Materials</a>. Other details are as those in <a href="#ijms-25-05429-f002" class="html-fig">Figure 2</a>. Full article ">Figure 6
Effect of PipX point mutations (*) on self- and cross-interactions with GlnB or GlnK in the BACTH system. (A) Self-interactions; (B) cross-interactions. Additional data are provided in <a href="#app1-ijms-25-05429" class="html-app">Figure S2 in the Supplementary Materials</a>. Other details are as those in <a href="#ijms-25-05429-f002" class="html-fig">Figure 2</a>. Full article ">Figure 7
Effects of PipX point mutations (*) on protein levels in E. coli. Top: representative immunodetection of PipX*, from CK1X or CK1XY constructions. Bottom: quantification of PipX* band intensities normalised by an unspecific band and referred to WT PipX levels. Data are presented as means and error bars (standard deviation) of four biological replicates. Full article ">

17 pages, 2284 KiB

Open AccessArticle

Analysing the Cyanobacterial PipX Interaction Network Using NanoBiT Complementation in Synechococcus elongatus PCC7942

by Carmen Jerez, Antonio Llop, Paloma Salinas, Sirine Bibak, Karl Forchhammer and Asunción Contreras

Int. J. Mol. Sci. 2024, 25(9), 4702; https://doi.org/10.3390/ijms25094702 - 25 Apr 2024

Cited by 2 | Viewed by 1606

Abstract

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding [...] Read more.

The conserved cyanobacterial protein PipX is part of a complex interaction network with regulators involved in essential processes that include metabolic homeostasis and ribosome assembly. Because PipX interactions depend on the relative levels of their different partners and of the effector molecules binding to them, in vivo studies are required to understand the physiological significance and contribution of environmental factors to the regulation of PipX complexes. Here, we have used the NanoBiT complementation system to analyse the regulation of complex formation in Synechococcus elongatus PCC 7942 between PipX and each of its two best-characterized partners, PII and NtcA. Our results confirm previous in vitro analyses on the regulation of PipX-PII and PipX-NtcA complexes by 2-oxoglutarate and on the regulation of PipX-PII by the ATP/ADP ratio, showing the disruption of PipX-NtcA complexes due to increased levels of ADP-bound PII in Synechococcus elongatus. The demonstration of a positive role of PII on PipX-NtcA complexes during their initial response to nitrogen starvation or the impact of a PipX point mutation on the activity of PipX-PII and PipX-NtcA reporters are further indications of the sensitivity of the system. This study reveals additional regulatory complexities in the PipX interaction network, opening a path for future research on cyanobacteria. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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Figure 1
NanoBiT constructs and strategy used to analyse the PipX-PII and PipX-NtcA interactions in S. elongatus. (A) The NSI region and derivatives containing the C.S3 selection marker and the corresponding gene fusions are schematically illustrated, with the relevant products depicted to the right. * refers to PipX or PipXY6A. (B) Schematic representation of the pipX and glnB alleles. (C) Left panel: PCR analysis indicating the primers, depicted as black arrows, in (A,B) and the size of bands at the left and right, respectively. M: λ EcoRI/HindIII size marker. Right panel: strains analysed. See text for additional details. Full article ">Figure 2
Levels of PipX and PII derivatives in S. elongatus. Representative immunodetection of PipX and PII of S. elongatus strains differing in their NSI constructs or genetic background (in brackets) as indicated. Relative PipX and PII levels were normalized by the PlmA signal and referred to the WT. Data are presented as means and error bars (standard deviation) from three biological replicates. Full article ">Figure 3
Real-time responses of PipX-PII and PipX-NtcA reporters to changes in energy levels and nitrogen sources. Reporter strains are indicated, and relevant proteins illustrated on top of the corresponding results. (A) Bioluminescence signals (black scale and curves) and normalized ATP levels (red scale and curves) from cultures grown with BG11 in the presence or absence of 200 µM DCCD. (B) Bioluminescence signals after transfer to the indicated nitrogen regimens. Data in (A,B) are presented as means with error bars (standard deviation) due to the indicated number of biological replicates (top rectangles) performed in each case. Wilcoxon rank-sum tests between the indicated comparisons produced p-values < 0.05 (*). (a–c) refer to the indicated nitrogen conditions. Full article ">Figure 4
Regulation of PipX levels and PipX-NtcA interactions by PII in response to nitrogen deprivation. (A) Representative immunodetection and relative levels of PipX (PipX) from S. elongatus cultures after two centrifuge/washing steps with BG110 (-N), BG11 (Fresh) or the same BG11 supernatant (Used), normalised to the intensity shown in the same blot by endogenous PlmA, and respective to the “Used” values. Data are presented as means and error bars (standard deviation) from five biological replicates of two independent experiments. Wilcoxon rank-sum test produced p-values < 0.05 (*) (B) Real-time comparison of bioluminescence signals under the indicated nitrogen regimens at different times between the pipX (Δ) and pipXglnB (○) strains. Data are presented as means and error bars (standard deviation) from two biological replicates. Other details as in <a href="#ijms-25-04702-f003" class="html-fig">Figure 3</a>. Full article ">Figure 5
Impact of the mutation Y6A on PipX-PII and PipX-NtcA interactions. Bioluminescence signal of the indicated strains under different nitrogen regimen conditions at timepoints 0 and 60′. Black, grey, and red lines correspond to the WT or Y6A versions of the reporter and to the PipX control, respectively. Data are presented as means and error bars (standard deviation) from the indicated biological replicates (squares inside the graphics). Inset covers enlarge their corresponding regions. Full article ">

14 pages, 1725 KiB

Open AccessArticle

In Vivo Detection of Metabolic Fluctuations in Real Time Using the NanoBiT Technology Based on PII Signalling Protein Interactions

by Rokhsareh Rozbeh and Karl Forchhammer

Int. J. Mol. Sci. 2024, 25(6), 3409; https://doi.org/10.3390/ijms25063409 - 17 Mar 2024

Cited by 2 | Viewed by 1622

Abstract

New protein-fragment complementation assays (PCA) have successfully been developed to characterize protein–protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, and straightforward read out. Previously, we [...] Read more.

New protein-fragment complementation assays (PCA) have successfully been developed to characterize protein–protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, and straightforward read out. Previously, we explored the in vitro protein interaction dynamics of the PII signalling protein using NanoBiT, revealing significant modulation of luminescence signals generated by the interaction between PII and its receptor protein NAGK by 2-oxoglutarate levels. In the current work, we investigated this technology in vivo, to find out whether recombinantly expressed NanoBiT constructs using the NanoLuc large fragment fused to PII and PII-interaction partners NAGK or PipX-fused to the NanoLuc Small BiT are capable of detecting the metabolic fluctuations in Escherichia coli. Therefore, we devised an assay capable of capturing the metabolic responses of E. coli cells, demonstrating real-time metabolic perturbation upon nitrogen upshift or depletion treatments. In particular, the PII-NAGK NanoBitT sensor pair reported these changes in a highly sensitive manner. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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11 pages, 2417 KiB

Open AccessCommunication

Structured Tandem Repeats in Protein Interactions

by Juan Mac Donagh, Abril Marchesini, Agostina Spiga, Maximiliano José Fallico, Paula Nazarena Arrías, Alexander Miguel Monzon, Aimilia-Christina Vagiona, Mariane Gonçalves-Kulik, Pablo Mier and Miguel A. Andrade-Navarro

Int. J. Mol. Sci. 2024, 25(5), 2994; https://doi.org/10.3390/ijms25052994 - 5 Mar 2024

Cited by 1 | Viewed by 1627

Abstract

Tandem repeats (TRs) in protein sequences are consecutive, highly similar sequence motifs. Some types of TRs fold into structural units that pack together in ensembles, forming either an (open) elongated domain or a (closed) propeller, where the last unit of the ensemble packs [...] Read more.

Tandem repeats (TRs) in protein sequences are consecutive, highly similar sequence motifs. Some types of TRs fold into structural units that pack together in ensembles, forming either an (open) elongated domain or a (closed) propeller, where the last unit of the ensemble packs against the first one. Here, we examine TR proteins (TRPs) to see how their sequence, structure, and evolutionary properties favor them for a function as mediators of protein interactions. Our observations suggest that TRPs bind other proteins using large, structured surfaces like globular domains; in particular, open-structured TR ensembles are favored by flexible termini and the possibility to tightly coil against their targets. While, intuitively, open ensembles of TRs seem prone to evolve due to their potential to accommodate insertions and deletions of units, these evolutionary events are unexpectedly rare, suggesting that they are advantageous for the emergence of the ancestral sequence but are early fixed. We hypothesize that their flexibility makes it easier for further proteins to adapt to interact with them, which would explain their large number of protein interactions. We provide insight into the properties of open TR ensembles, which make them scaffolds for alternative protein complexes to organize genes, RNA and proteins. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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Figure 1
Properties of TRs by type and compared to other protein regions. (a) Number of repeat units identified. HEAT_AAA, HEAT_ADB and HEAT_IMB are three variants of HEAT repeats and can be redundant. TRs forming open and closed ensembles are indicated with black and red labels, respectively. (b) Average number of protein partners for TRPs. Horizontal dotted lines indicate the values for all human proteins (proteome), for proteins annotated with globular domains (Pfam), and for all TRPs. (c) Frequency of amino acids in SLiMs. Values are shown for: the complete human proteome (All), for proteins that do not contain TRs (non TRPs), for TRPs, for residues in TRs of TRPs (TRPs: in TRs), for residues outside TRs in TRPs (TRPs: out TRs), and in annotated globular domains (Pfam). (d) Frequency of amino acids in phosphorylation sites. (e) Frequency of amino acids in IDRs. (f) Disordered content by repeat type (PFTA and PFTB are not displayed because their numbers are too low). To prepare the plots (c–f), we obtained the coordinates of the features (SLiMs, phosphorylation sites, disorder regions) in all human sequences from the corresponding databases (see Methods for details) and then divided the number of residues within the given feature by the total of residues in the corresponding type of sequence. Full article ">Figure 2
pLDDT scores of AlphaFold predictions along ensembles of TRs. (a) WD40 (closed ensemble) compared to an average of four open ensembles (shown in (b)). (b) Values for the four open ensembles. The x-axis indicates the relative position in the TR ensemble N- to C-terminal. Full article ">Figure 3
Flexibility of interacting TRPs. Structures of protein complexes with TRPs by repeat type. Elongated: ARM repeats in human catenin beta-1 binding NR5A2 (PDB:3TX7); LRR repeats in Toll-like receptor 4 binding LY96 (PDB:4G8A); HEAT repeats in importin subunit beta-1 shown in the same orientation, forming three complexes with histone H1.0 (PDB:6N88), Zinc finger protein SNAI1 (PDB:3W5K) and the IBB domain of Snurportin-1 (PDB:2QNA). Cyclic: RCC1 in RPGR repeats binding the interacting domain of RPGRIP1 (PDB:4QAM) and KELCH repeats in ARPC1B binding ARPC4 (PDB:6YW6). TRP in purple and bound protein in yellow. Full article ">Figure 4
Cases of fast evolution in Plasmodium species. (a) Gain of an LRR unit in A0A1D3JKH0_PLAMA positions 595–618. (b) Gain of a TPR unit in A0A1D3TFE6_PLAMA positions 660–693 (colored in red in the structure). Sequence identifiers from UniProtKB. In order, species are: Plasmodium berghei, Plasmodium relictum, Plasmodium malariae, Plasmodium knowlesi, Plasmodium gonderi and Plasmodium falciparum. No alternative spliced isoforms for A0A1D3JKH0_PLAMA or A0A1D3TFE6_PLAMA are given in UniProt (February 2024). The structures shown are models from AlphaFold [<a href="#B41-ijms-25-02994" class="html-bibr">41</a>] and Robetta [<a href="#B42-ijms-25-02994" class="html-bibr">42</a>] (left and right, respectively). Full article ">Figure 4 Cont.
Cases of fast evolution in Plasmodium species. (a) Gain of an LRR unit in A0A1D3JKH0_PLAMA positions 595–618. (b) Gain of a TPR unit in A0A1D3TFE6_PLAMA positions 660–693 (colored in red in the structure). Sequence identifiers from UniProtKB. In order, species are: Plasmodium berghei, Plasmodium relictum, Plasmodium malariae, Plasmodium knowlesi, Plasmodium gonderi and Plasmodium falciparum. No alternative spliced isoforms for A0A1D3JKH0_PLAMA or A0A1D3TFE6_PLAMA are given in UniProt (February 2024). The structures shown are models from AlphaFold [<a href="#B41-ijms-25-02994" class="html-bibr">41</a>] and Robetta [<a href="#B42-ijms-25-02994" class="html-bibr">42</a>] (left and right, respectively). Full article ">Figure 5
Functional enrichment of TRP interactors. Top: Biological Process. Middle: Cellular Component. Bottom: Molecular Function. Gene Ontology (GO) enrichment analysis was carried out for a set of TRP interactors (All) and then separately for the interactors of each TRP type (see Methods for details). Enriched GO Biological Process (BP), Molecular Function (MF) and Cellular Component (CC) terms with the lowest adjusted p-value were kept. Full article ">

22 pages, 3659 KiB

Open AccessArticle

Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein–Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System

by Sára Ferková, Ulrike Froehlich, Marie-Édith Nepveu-Traversy, Alexandre Murza, Taha Azad, Michel Grandbois, Philippe Sarret, Pierre Lavigne and Pierre-Luc Boudreault

Int. J. Mol. Sci. 2024, 25(1), 166; https://doi.org/10.3390/ijms25010166 - 21 Dec 2023

Viewed by 1840

Abstract

Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled [...] Read more.

Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints—lactam, hydrocarbon, triazole, double triazole and thioether—on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2). Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Staple screening performed on a short hACE2-derived α-helical sequence (His34-Gln42) (shown in pine green) via i, i + 4 side-to-side chain cyclizations by lactamization, olefin ring-closing metathesis (RCM), S-alkylation, S-arylation and copper(I)-catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition (CuAAC). Ball-and-stick model staple representations are shown in the insets and macrocycle sizes are indicated in the upper-righthand corner. Residues from hACE2 involved in the SARS-CoV-2 S RBD/hACE2 interaction are shown as yellow sticks labeled with three-letter codes. Full article ">Figure 2
In silico alanine scan (BudeAlaScan a) results for α1-helix (Ser19-Thr52) of hACE2 taken from the crystal structure of SARS-CoV-2 S RBD bound with hACE2 (PDB 6M0J). Residues from hACE2 involved in the SARS-CoV-2 S RBD/hACE2 interface are shown as yellow sticks labeled with three-letter codes. Selected hACE2-derived sequences (Asp30-Asp38) and (His34-Gln42) are outlined in light green and blue, respectively. a BudeAlaScan is an online software (version 1.0) available at <a href="https://pragmaticproteindesign.bio.ed.ac.uk/balas/" target="_blank">https://pragmaticproteindesign.bio.ed.ac.uk/balas/</a> (accessed on 7 June 2023); it is only applicable to proteins consisting of natural amino acids. Full article ">Figure 3
Data generated from Far-UV CD spectra for hACE2-derived peptides measured at a concentration of 100 µM in 10 mM sodium phosphate buffer at pH 7.4 and 25 °C. (A) Far-UV CD spectra of hACE2 (Asp30-Asp38)-derived linear and i, i + 4 staple peptides in molar ellipticity per residue. (B) Table listing the derivatives of 1 with a focus on the macrocyclization technique and measure-based calculated helicity (%) via CD. (C) CD spectra of hACE2 (His34-Gln42)-derived linear and i, i + 4 staple peptides in molar ellipticity per residue. (D) Table listing the derivatives of 2 with a focus on the macrocyclization technique and measure-based calculated helicity (%) via CD. HDY 1,5-Hexadiyne; BMB 1,4-Bis(bromomethyl)benzene; HFB hexafluorobenzene. a Predicted helicity (%) using AGADIR online software available at <a href="http://agadir.crg.es" target="_blank">http://agadir.crg.es</a> (accessed on 17 April 2023); it is only applicable to peptides consisting of natural amino acids. Full article ">Figure 4
Secondary chemical shifts a of (A) CαH and (B) NH protons for peptide 4. Secondary chemical shifts of linker-formed residues have been highlighted with an orange-colored outline. a Random-coil chemical shifts for 20 common amino acids followed by alanine were measured using a peptide with free N- and C-termini at pH 5.0 and 25 °C. Full article ">Figure 5
Characteristic short- and medium-range sequential NOEs for an α-helix assessment. (A) The dNN and (B) dαN regions of a NOESY spectrum recorded for peptide 4. (C) Graphical illustration of sequential and medium-range 1H-1H distances in a peptide sequence. (D) Schematic representation of NOESY patterns involving NH and CαH protons observed in a NOESY spectrum recorded for α-helix compared with peptide 4. The horizontal lines of various lengths indicate NOE connectivities between protons of peptide sequences; the thicknesses of the lines is proportional to the observed strong, medium, and weak NOEs signal intensities. * The protons could not be assigned unambiguously. Full article ">Figure 6
The SARS-CoV-2 Spike RBD/hACE2 inhibition assessment involving a bioluminescence-based bioreporter assay and a pseudovirus-based entry inhibition assay. (A) Split-luciferase bioreporter assay demonstrating the disruptor capacity of peptides 1, 2, linear 4, 4, linear 11 and 11. Asterisks indicate a statistically significant difference between the RLUs measured for SmBiT-ACE2 + LgBiT-RBD and the neutralizing Ab control or an individual peptide. (B) Antiviral activity of peptides linear 4, 4, linear 11 and 11 was assessed using a SARS-CoV-2 pseudovirus carrying a fluorescent reporter gene and HEK-293T-hACE2 cells transfected with TMPRSS2. The fluorescence data (RFUs) were converted into percentages via normalization with the infection-free control (Dulbecco’s Eagle Medium; DMEM) set as 0%; we furthermore considered that the pseudovirus-only samples represented maximum infection (100%) using GraphPad Prism software (version 9.3.1). Both experiments were repeated in triplicate three times, and the data are expressed as means ± standard error of the mean (SEM) (error bars). The means of more than two groups were compared using one-way ANOVA with Tukey’s multiple comparison correction. For all analyses, **** p < 0.0001; ** 0.0017 <math display="inline"><semantics> <mrow> <mo>≤</mo> </mrow> </semantics></math> p <math display="inline"><semantics> <mrow> <mo>≤</mo> </mrow> </semantics></math> 0.0025; n.s., not significant. Full article ">Figure 7
Plasma stability measured in rat plasma over 24 h of incubation at 37 °C. (A) The proteolytic stability of hACE2 (Asp30-Asp38)-derived peptides recorded as a function of degraded peptide over 24 h. (B) The proteolytic stability of hACE2 (His34-Gln42)-derived peptides recorded as a function of degraded peptide over 24 h. The data are plotted as means and SEMs of duplicate independent experiments. The percentage of residual peptide was monitored using UPLC-MS. All of the experiments were repeated three times. Full article ">

21 pages, 17776 KiB

Open AccessArticle

Inhibiting Intracellular α_2C-Adrenoceptor Surface Translocation Using Decoy Peptides: Identification of an Essential Role of the C-Terminus in Receptor Trafficking

by Aisha Raza, Saima Mohsin, Fasiha Saeed, Syed Abid Ali and Maqsood A. Chotani

Int. J. Mol. Sci. 2023, 24(24), 17558; https://doi.org/10.3390/ijms242417558 - 16 Dec 2023

Viewed by 1652

Abstract

The G protein-coupled α₂-adrenoceptor subtype C (abbreviated α_2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet–hands, Raynaud’s phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α_2C-adrenoceptors are expressed in specialized smooth [...] Read more.

The G protein-coupled α₂-adrenoceptor subtype C (abbreviated α_2C-AR) has been implicated in peripheral vascular conditions and diseases such as cold feet–hands, Raynaud’s phenomenon, and scleroderma, contributing to morbidity and mortality. Microvascular α_2C-adrenoceptors are expressed in specialized smooth muscle cells and mediate constriction under physiological conditions and the occlusion of blood supply involving vasospastic episodes and tissue damage under pathological conditions. A crucial step for receptor biological activity is the cell surface trafficking of intracellular receptors, triggered by cAMP-Epac-Rap1A GTPase signaling, which involves protein–protein association with the actin-binding protein filamin-2, mediated by critical amino acid residues in the last 14 amino acids of the receptor carboxyl (C)-terminus. This study assessed the role of the C-terminus in Rap1A GTPase coupled receptor trafficking by domain-swapping studies using recombinant tagged receptors in transient co-transfections and compared with wild-type receptors using immunofluorescence microscopy. We further tested the biological relevance of the α_2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α_2C-AR surface translocation in transfected as well as in microvascular smooth muscle cells expressing endogenous receptors. These studies contribute to establishing proof of principle to target intracellular α_2C-adrenoceptors to reduce biological activity, which in clinical conditions can be a target for therapy. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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20 pages, 2516 KiB

Open AccessArticle

Endoplasmic Reticulum Protein TXNDC5 Interacts with PRDX6 and HSPA9 to Regulate Glutathione Metabolism and Lipid Peroxidation in the Hepatic AML12 Cell Line

by Seyed Hesamoddin Bidooki, Javier Sánchez-Marco, Roberto Martínez-Beamonte, Tania Herrero-Continente, María A. Navarro, María J. Rodríguez-Yoldi and Jesús Osada

Int. J. Mol. Sci. 2023, 24(24), 17131; https://doi.org/10.3390/ijms242417131 - 5 Dec 2023

Cited by 4 | Viewed by 2182

Abstract

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded [...] Read more.

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded protein response. The ER is the primary location site for chaperones like thioredoxin domain-containing 5 (TXNDC5). Glutathione participates in cellular oxidative stress, and its interaction with TXNDC5 in the ER may decrease the disulfide bonds of this protein. In addition, glutathione is utilized by glutathione peroxidases to inactivate oxidized lipids. To characterize proteins interacting with TXNDC5, immunoprecipitation and liquid chromatography–mass spectrometry were used. Lipid peroxidation, reduced glutathione, inducible phospholipase A₂ (iPLA₂) and hepatic transcriptome were assessed in the AML12 and TXNDC5-deficient AML12 cell lines. The results showed that HSPA9 and PRDX6 interact with TXNDC5 in AML12 cells. In addition, TXNDC5 deficiency reduced the protein levels of PRDX6 and HSPA9 in AML12. Moreover, lipid peroxidation, glutathione and iPLA₂ activities were significantly decreased in TXNDC5-deficient cells, and to find the cause of the PRDX6 protein reduction, proteasome suppression revealed no considerable effect on it. Finally, hepatic transcripts connected to PRDX6 and HSPA9 indicated an increase in the Dnaja3, Mfn2 and Prdx5 and a decrease in Npm1, Oplah, Gstp3, Gstm6, Gstt1, Serpina1a, Serpina1b, Serpina3m, Hsp90aa1 and Rps14 mRNA levels in AML12 KO cells. In conclusion, the lipid peroxidation system and glutathione mechanism in AML12 cells may be disrupted by the absence of TXNDC5, a novel protein–protein interacting partner of PRDX6 and HSPA9. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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Figure 1

Figure 1
Confirmation of proteins interacting with TXNDC5 observed via Co-IP and mass spectrometry. Co-immunoprecipitation assays of AML12 WT and KO were immunoprecipitated with the TXNDC5 antibody, and the precipitates were analyzed by means of Western blot using an antibody against (A) TXNDC5 and (B) HSPA9. (C) AML12 WT and KO were co-immunoprecipitated with the PRDX6 antibody, and (D) the precipitates were examined via Western blotting with the anti-TXNDC5 antibody. Description of the lanes from left to right (A–C): first lane: co-immunoprecipitation in KO AML12 cells sample, second lane: co-immunoprecipitation in WT AML12 cells sample, third lane: WT AML12 cells sample without co-immunoprecipitation as input. (D) First and third lanes: co-immunoprecipitation in WT AML12 cells sample, second lane: co-immunoprecipitation in KO AML12 cells sample; fourth lane: WT AML12 cells sample without co-immunoprecipitation as input. Full article ">Figure 2
(A,B) Expression pattern of HSPA9. mRNA level of Hspa9 in the (A) AML12 cell line. (B) Protein level of HSPA9 and its Western blot in the hepatic cell line. (C–E) Expression pattern of PRDX6. The RNA expression level of Prdx6 (C) in the AML12 cell line and (D) the mRNA level of Prdx6b is shown in the AML12 cell line. (E) The protein level of PRDX6 was analyzed using Western blot in the hepatic cell lines. The Mann–Whitney U test for pairwise comparisons was used for statistical analysis; * p < 0.05, ** p < 0.01. (F) Effect of a proteasome inhibitor on the PRDX6 protein levels in the AML12 WT and KO cell lines. Cells were exposed to 5 and 10 µM MG-132 as a proteasome inhibitor for 24 h; then, the proteins were extracted and analyzed via Western blot. The MG-132-exposed WT cells displayed the same PRDX6 bands as the control group; even so, the KO cells did not show any bands. (HSC70 was used as a control protein.) Full article ">Figure 3
(A) Lipid peroxidation analysis. Significant MDA reduction in the KO cells is shown. (B–D) Assessment of glutathione and iPLA2 activities. Considerable decrements of (B) GSH and (C) iPLA2 activity in the KO cells are indicated. (D) AML12 KO cells display a remarkable increment in the total PLA2. Statistical analysis was carried out according to the Mann–Whitney U test for pairwise comparisons; * p < 0.05, *** p < 0.001. Full article ">Figure 4
(A–C) Possible protein–protein interactions of mouse (A) TXNDC5, (B) PRDX6 and (C) HSPA9 generated by the String database. High confidence of 0.7 and not more than 20 interactors are shown. Nodes: network nodes represent proteins; red nodes: query proteins, colored nodes: the first shell of interactors, white nodes: the second shell of interactors. Edges represent protein–protein associations; blue and purple edges: known interactions, green, red and dark-blue edges: predicted interactions, yellow, black and light-blue: others. (D) Identification algorithm of genes associated with PRDX6 and HSPA9 based on the PubMed, KEGG, String, Mouse Genome Informatics, and Alliance of Genome Resource databases. Identification was based on mRNA changes according to the RNAseq analysis of the AML12 WT and KO cell lines (SL2R > ±2 in the Prdx6 dataset and SL2R > ±1 in the Hspa9 dataset). The liver protein abundance (parts per million (ppm)) based on the PAX database was considered as a final step (PPM > 300 in the Prdx6 dataset and PPM > 100 in the Hspa9 dataset). Expression of (E) Hspa9- and (F) Prdx6-associated genes in the AML12 cell line. Data are mean ± SD. Statistical analysis was performed according to the Mann–Whitney U test. * p < 0.05, ** p < 0.01. Full article ">Figure 5
Diagram illustrating the interactions of the TXNDC5 protein with HSPA9 and PRDX6 as well as the impact of TXNDC5 deficiency in AML12 KO cells on different transcriptomes and their functions. Microsoft Publisher Document Version 2010 was used to create this schematic. ↑ Significantly increased, ↓ Significantly decreased. Red circle and cross: Elimination. Full article ">

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NF2-Related Schwannomatosis (NF2): Molecular Insights and Therapeutic Avenues

by Bae-Hoon Kim, Yeon-Ho Chung, Tae-Gyun Woo, So-mi Kang, Soyoung Park, Minju Kim and Bum-Joon Park

Int. J. Mol. Sci. 2024, 25(12), 6558; https://doi.org/10.3390/ijms25126558 - 14 Jun 2024

Cited by 1 | Viewed by 2488

Abstract

NF2-related schwannomatosis (NF2) is a genetic syndrome characterized by the growth of benign tumors in the nervous system, particularly bilateral vestibular schwannomas, meningiomas, and ependymomas. This review consolidates the current knowledge on NF2 syndrome, emphasizing the molecular pathology associated with the mutations in [...] Read more.

NF2-related schwannomatosis (NF2) is a genetic syndrome characterized by the growth of benign tumors in the nervous system, particularly bilateral vestibular schwannomas, meningiomas, and ependymomas. This review consolidates the current knowledge on NF2 syndrome, emphasizing the molecular pathology associated with the mutations in the gene of the same name, the NF2 gene, and the subsequent dysfunction of its product, the Merlin protein. Merlin, a tumor suppressor, integrates multiple signaling pathways that regulate cell contact, proliferation, and motility, thereby influencing tumor growth. The loss of Merlin disrupts these pathways, leading to tumorigenesis. We discuss the roles of another two proteins potentially associated with NF2 deficiency as well as Merlin: Yes-associated protein 1 (YAP), which may promote tumor growth, and Raf kinase inhibitory protein (RKIP), which appears to suppress tumor development. Additionally, this review discusses the efficacy of various treatments, such as molecular therapies that target specific pathways or inhibit neomorphic protein–protein interaction caused by NF2 deficiency. This overview not only expands on the fundamental understanding of NF2 pathophysiology but also explores the potential of novel therapeutic targets that affect the clinical approach to NF2 syndrome. Full article

(This article belongs to the Special Issue Advances in Protein-Protein Interactions—2nd Edition)

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