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Advances in Protein-Protein Interactions—2nd Edition

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pharmacology".

Deadline for manuscript submissions: closed (31 July 2024) | Viewed by 14831

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Guest Editor
Biosciences and Food Technology, School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, VIC 3053, Australia
Interests: drug discovery; protein-protein interactions; structural bioinformatics; molecular modelling
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

There are over 300,000 protein–protein interaction (PPI) pairs identified in the human genome. Thus, it is not surprising that modulators of PPIs—ideally small “drug-like” molecules—are urgently being sought and developed by the pharmaceutical industry to address the unmet medical needs. However, the physical characteristics of the PPI interface make this task non-trivial. Furthermore, unlike the traditional pharmaceutical approach of focusing on finding a ‘single switch that works’, it is clear that the phenotype of many diseases relies on complex networks of PPIs. Destabilising these networks for a successful therapeutic approach will require perturbing multiple key interactions.

This Special Issue focuses on recent studies aiming to investigate protien–protein interactions, with an additional aim of developing drugs to modulate these interactions. Specifically, this Special Issue will explore the latest computational and structural biology methods, and studies that further our understanding of protein– protein interfaces and how to better develop molecules to modulate these are welcomed.

Dr. Jessica Holien
Guest Editor

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Keywords

  • protein–protein interactions
  • structural biology
  • bioinformatics
  • network analysis
  • drug design
  • drug development
  • computational drug design
  • protein–ligand interactions
  • target identification

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Related Special Issue

Published Papers (8 papers)

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17 pages, 1230 KiB  
Article
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 1110
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

Figure 1
<p>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.</p>
Full article ">Figure 2
<p>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.</p>
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<p>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.</p>
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<p>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>.</p>
Full article ">Figure 5
<p>PII gives weaker self- and cross-interaction signals than GlnB or GlnK in the BACTH system. T25 derivatives were expressed from pKT25 vector (<b>A</b>) or pT25 vector (<b>B</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>.</p>
Full article ">Figure 6
<p>Effect of PipX point mutations (*) on self- and cross-interactions with GlnB or GlnK in the BACTH system. (<b>A</b>) Self-interactions; (<b>B</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>.</p>
Full article ">Figure 7
<p>Effects of PipX point mutations (*) on protein levels in <span class="html-italic">E. coli</span>. 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.</p>
Full article ">
17 pages, 2284 KiB  
Article
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 1594
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
<p>NanoBiT constructs and strategy used to analyse the PipX-PII and PipX-NtcA interactions in <span class="html-italic">S. elongatus</span>. (<b>A</b>) 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 PipX<sup>Y6A</sup>. (<b>B</b>) Schematic representation of the <span class="html-italic">pipX</span> and <span class="html-italic">glnB</span> alleles. (<b>C</b>) <b>Left</b> panel: PCR analysis indicating the primers, depicted as black arrows, in (<b>A</b>,<b>B</b>) and the size of bands at the left and right, respectively. M: λ EcoRI/HindIII size marker. <b>Right</b> panel: strains analysed. See text for additional details.</p>
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<p>Levels of PipX and PII derivatives in <span class="html-italic">S. elongatus</span>. Representative immunodetection of PipX and PII of <span class="html-italic">S. elongatus</span> 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.</p>
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<p>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. (<b>A</b>) 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>B</b>) Bioluminescence signals after transfer to the indicated nitrogen regimens. Data in (<b>A</b>,<b>B</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 <span class="html-italic">p</span>-values &lt; 0.05 (*). (a–c) refer to the indicated nitrogen conditions.</p>
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<p>Regulation of PipX levels and PipX-NtcA interactions by PII in response to nitrogen deprivation. (<b>A</b>) Representative immunodetection and relative levels of PipX (PipX) from <span class="html-italic">S. elongatus</span> cultures after two centrifuge/washing steps with BG11<sub>0</sub> (-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 <span class="html-italic">p</span>-values &lt; 0.05 (*) (<b>B</b>) Real-time comparison of bioluminescence signals under the indicated nitrogen regimens at different times between the <span class="html-italic">pipX</span> (Δ) and <span class="html-italic">pipXglnB</span> (○) 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>.</p>
Full article ">Figure 5
<p>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.</p>
Full article ">
14 pages, 1725 KiB  
Article
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 1610
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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Figure 1
<p>Expression vectors and fusion protein constructs. Schematic overview of PII-LgBiT—NAGK-SmBiT, PII-LgBiT—PipX-SmBiT, PII-Full length (FL), PII (S49E)-LgBiT—NAGK-SmBiT and PII-LgBiT proteins. Flexible linker with 8 amino acids (8aa) fused to LgBiT and flexible linker with 16 amino acids (16aa) fused to SmBiT in constructs.</p>
Full article ">Figure 1 Cont.
<p>Expression vectors and fusion protein constructs. Schematic overview of PII-LgBiT—NAGK-SmBiT, PII-LgBiT—PipX-SmBiT, PII-Full length (FL), PII (S49E)-LgBiT—NAGK-SmBiT and PII-LgBiT proteins. Flexible linker with 8 amino acids (8aa) fused to LgBiT and flexible linker with 16 amino acids (16aa) fused to SmBiT in constructs.</p>
Full article ">Figure 2
<p>Sensitivity of luminescence intensity (in RLU) in the experimental set-up towards oxygen consumption during the time-course experiments. (<b>A</b>) Control measurement using the PII-FL reporter. Measurement was briefly interrupted at time 120 s and 370 s and the samples were shaken and directly measured again. (<b>B</b>) As in part (<b>A</b>) but using the PII-LgBiT—NAGK-SmBiT sensor; here, the cells were shaken at 80 s and 320 s.</p>
Full article ">Figure 3
<p>Luminescence response of the PII-NAGK NanoBiT sensor towards ammonium upshift treatments (<b>A</b>,<b>B</b>) and as control, of the PII-FL reporter under identical test conditions (<b>C</b>,<b>D</b>). (<b>A</b>) Time course of the luminescence signal (RLU) after addition of luminescence reagent in untreated sample (1 mM NH<sub>4</sub>Cl), and to samples, where the NH<sub>4</sub>Cl concentration was increased to 4 mM or 40 mM. (<b>B</b>) Normalization of the RLU response curve to the RLU response curve of the reference sample (untreated, 1 mM NH<sub>4</sub>Cl) (<b>C</b>,<b>D</b>): as part (<b>A</b>,<b>B</b>) but using the PII-FL reporter for comparison. Average values from three measurements are shown and error bars are removed for a better comparison. The average standard deviation (STD) of each of the three replicates of the PII-NAGK sensor for ammonium treatments was less than 23%. The average standard deviation (STD) of each of the three replicates of the PII-FL sensor for ammonium treatments was less than 14%.</p>
Full article ">Figure 4
<p>Luminescence response of cells carrying the PII-NAGK NanoBiT sensor following inhibition of ammonium assimilation by methionine sulfoximine (MSX) or following nitrogen starvation. For comparison, the normalized RLU curves of the PII-FL reporter subjected to the same treatments are shown (<b>E</b>,<b>F</b>). (<b>A</b>) RLU time-course measurement of the PII-NAGK NanoBiT sensor in the absence and presence of 0.1 mM and 1 mM MSX. (<b>B</b>) Normalizing the RLU response curve to the untreated reference (0 MSX). (<b>C</b>) Normalized RLU response curve upon MSX treatment using the PII-FL reporter. (<b>D</b>) RLU time-course measurement following nitrogen depletion. (<b>E</b>) Normalizing the RLU response curves shown in (<b>D</b>) to the untreated reference. (<b>F</b>) Normalized RLU response curve upon nitrogen-deprivation using the PII-FL reporter. Average values from three measurements are shown and error bars are removed for a better comparison. The average standard deviation (STD) of each of the three replicates of the PII-NAGK sensor for inhibition of ammonium assimilation by MSX and nitrogen starvation was less than 25%. The average standard deviation (STD) of each of the three replicates of the PII-FL sensor for inhibition of ammonium assimilation by MSX was less than 15%. The average standard deviation (STD) of each of the three replicates of the PII-FL sensor for nitrogen starvation was less than 13%.</p>
Full article ">Figure 4 Cont.
<p>Luminescence response of cells carrying the PII-NAGK NanoBiT sensor following inhibition of ammonium assimilation by methionine sulfoximine (MSX) or following nitrogen starvation. For comparison, the normalized RLU curves of the PII-FL reporter subjected to the same treatments are shown (<b>E</b>,<b>F</b>). (<b>A</b>) RLU time-course measurement of the PII-NAGK NanoBiT sensor in the absence and presence of 0.1 mM and 1 mM MSX. (<b>B</b>) Normalizing the RLU response curve to the untreated reference (0 MSX). (<b>C</b>) Normalized RLU response curve upon MSX treatment using the PII-FL reporter. (<b>D</b>) RLU time-course measurement following nitrogen depletion. (<b>E</b>) Normalizing the RLU response curves shown in (<b>D</b>) to the untreated reference. (<b>F</b>) Normalized RLU response curve upon nitrogen-deprivation using the PII-FL reporter. Average values from three measurements are shown and error bars are removed for a better comparison. The average standard deviation (STD) of each of the three replicates of the PII-NAGK sensor for inhibition of ammonium assimilation by MSX and nitrogen starvation was less than 25%. The average standard deviation (STD) of each of the three replicates of the PII-FL sensor for inhibition of ammonium assimilation by MSX was less than 15%. The average standard deviation (STD) of each of the three replicates of the PII-FL sensor for nitrogen starvation was less than 13%.</p>
Full article ">Figure 5
<p>Effect of ammonium addition, MSX treatment and nitrogen starvation on luminescence after addition of luminescence reagent to cells carrying the PII-LgBiT—PipX-SmBiT sensor construct. (<b>A</b>) Time course of the luminescence signal (RLU) in untreated sample (1 mM NH<sub>4</sub>Cl), and to samples, where the NH<sub>4</sub>Cl concentration was increased to 4 mM or 40 mM. (<b>B</b>) Normalizing the RLU response curve of PII-PipX sensor to the reference sample. (<b>C</b>) RLU time course of PII-PipX NanoBiT sensor in the absence and presence of 0.1 mM and 1 mM MSX. (<b>D</b>) RLU response curves of (<b>C</b>) normalized to the reference sample. (<b>E</b>) RLU time course of PII-PipX NanoBiT sensor following nitrogen depletion. (<b>F</b>) RLU response curves of (<b>E</b>) normalized to the reference sample. Average values from three measurements are shown and error bars are removed for a better comparison. The average standard deviation (STD) of each of the three replicates of the PII-PipX sensor for ammonium treatments was less than 21%. The average standard deviation (STD) of each of the three replicates of the PII-PipX sensor for inhibition of ammonium assimilation by MSX was less than 24%. The average standard deviation (STD) of each of the three replicates of the PII-PipX sensor for nitrogen starvation was less than 23%.</p>
Full article ">Figure 6
<p>Detection of the metabolic response of E. coli cells shifted from M9 medium into distilled water, using the PII-LgBiT—NAGK-SmBiT sensor or as a control the PII-FL-reporter. (<b>A</b>) RLU response curve from the PII-LgBiT—NAGK-SmBiT sensor of treated sample (red) and untreated reference sample (blue) (<b>B</b>) RLU response curves of (<b>A</b>) normalized to the reference sample. (<b>C</b>) RLU response curve from the PII-FL reporter of treated sample (red) and untreated reference sample (blue). (<b>D</b>) RLU response curves of (<b>C</b>) normalized to the reference sample. Average values from three measurements are shown and error bars are removed for a better comparison. The average standard deviation (STD) of each of the three replicates of the PII-NAGK sensor for nutrient deprivation in distilled water was less than 18%. The average standard deviation (STD) of each of the three replicates of the PII-FL sensor for nutrient deprivation in distilled water was less than 11%.</p>
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11 pages, 2417 KiB  
Communication
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 1614
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
<p>Properties of TRs by type and compared to other protein regions. (<b>a</b>) 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>b</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. (<b>c</b>) 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). (<b>d</b>) Frequency of amino acids in phosphorylation sites. (<b>e</b>) Frequency of amino acids in IDRs. (<b>f</b>) Disordered content by repeat type (PFTA and PFTB are not displayed because their numbers are too low). To prepare the plots (<b>c</b>–<b>f</b>), 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.</p>
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<p>pLDDT scores of AlphaFold predictions along ensembles of TRs. (<b>a</b>) WD40 (closed ensemble) compared to an average of four open ensembles (shown in (<b>b</b>)). (<b>b</b>) Values for the four open ensembles. The x-axis indicates the relative position in the TR ensemble N- to C-terminal.</p>
Full article ">Figure 3
<p>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.</p>
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<p>Cases of fast evolution in <span class="html-italic">Plasmodium</span> species. (<b>a</b>) Gain of an LRR unit in A0A1D3JKH0_PLAMA positions 595–618. (<b>b</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: <span class="html-italic">Plasmodium berghei</span>, <span class="html-italic">Plasmodium relictum</span>, <span class="html-italic">Plasmodium malariae</span>, <span class="html-italic">Plasmodium knowlesi</span>, <span class="html-italic">Plasmodium gonderi</span> and <span class="html-italic">Plasmodium falciparum</span>. 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).</p>
Full article ">Figure 4 Cont.
<p>Cases of fast evolution in <span class="html-italic">Plasmodium</span> species. (<b>a</b>) Gain of an LRR unit in A0A1D3JKH0_PLAMA positions 595–618. (<b>b</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: <span class="html-italic">Plasmodium berghei</span>, <span class="html-italic">Plasmodium relictum</span>, <span class="html-italic">Plasmodium malariae</span>, <span class="html-italic">Plasmodium knowlesi</span>, <span class="html-italic">Plasmodium gonderi</span> and <span class="html-italic">Plasmodium falciparum</span>. 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).</p>
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<p>Functional enrichment of TRP interactors. <b>Top</b>: Biological Process. <b>Middle</b>: Cellular Component. <b>Bottom</b>: 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 <span class="html-italic">p</span>-value were kept.</p>
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22 pages, 3659 KiB  
Article
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 1834
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)
Show Figures

Graphical abstract

Graphical abstract
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<p>Staple screening performed on a short hACE2-derived α-helical sequence (His34-Gln42) (shown in pine green) via <span class="html-italic">i</span>, <span class="html-italic">i</span> + 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.</p>
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<p>In silico alanine scan (BudeAlaScan <sup>a</sup>) 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. <sup>a</sup> 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.</p>
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<p>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. (<b>A</b>) Far-UV CD spectra of hACE2 (Asp30-Asp38)-derived linear and <span class="html-italic">i</span>, <span class="html-italic">i</span> + 4 staple peptides in molar ellipticity per residue. (<b>B</b>) Table listing the derivatives of <b>1</b> with a focus on the macrocyclization technique and measure-based calculated helicity (%) via CD. (<b>C</b>) CD spectra of hACE2 (His34-Gln42)-derived linear and <span class="html-italic">i</span>, <span class="html-italic">i</span> + 4 staple peptides in molar ellipticity per residue. (<b>D</b>) Table listing the derivatives of <b>2</b> 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. <sup>a</sup> 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.</p>
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<p>Secondary chemical shifts <sup>a</sup> of (<b>A</b>) CαH and (<b>B</b>) NH protons for peptide <b>4</b>. Secondary chemical shifts of linker-formed residues have been highlighted with an orange-colored outline. <sup>a</sup> 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.</p>
Full article ">Figure 5
<p>Characteristic short- and medium-range sequential NOEs for an α-helix assessment. (<b>A</b>) The d<sub>NN</sub> and (<b>B</b>) d<sub>αN</sub> regions of a NOESY spectrum recorded for peptide <b>4</b>. (<b>C</b>) Graphical illustration of sequential and medium-range <sup>1</sup>H-<sup>1</sup>H distances in a peptide sequence. (<b>D</b>) Schematic representation of NOESY patterns involving NH and CαH protons observed in a NOESY spectrum recorded for α-helix compared with peptide <b>4</b>. 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.</p>
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<p>The SARS-CoV-2 Spike RBD/hACE2 inhibition assessment involving a bioluminescence-based bioreporter assay and a pseudovirus-based entry inhibition assay. (<b>A</b>) Split-luciferase bioreporter assay demonstrating the disruptor capacity of peptides <b>1</b>, <b>2</b>, <b>linear 4</b>, <b>4</b>, <b>linear 11</b> and <b>11</b>. 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>B</b>) Antiviral activity of peptides <b>linear 4</b>, <b>4</b>, <b>linear 11</b> and <b>11</b> 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, **** <span class="html-italic">p</span> &lt; 0.0001; ** 0.0017 <math display="inline"><semantics> <mrow> <mo>≤</mo> </mrow> </semantics></math> <span class="html-italic">p</span> <math display="inline"><semantics> <mrow> <mo>≤</mo> </mrow> </semantics></math> 0.0025; n.s., not significant.</p>
Full article ">Figure 7
<p>Plasma stability measured in rat plasma over 24 h of incubation at 37 °C. (<b>A</b>) The proteolytic stability of hACE2 (Asp30-Asp38)-derived peptides recorded as a function of degraded peptide over 24 h. (<b>B</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.</p>
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21 pages, 17776 KiB  
Article
Inhibiting Intracellular α2C-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 1646
Abstract
The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-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 α2C-adrenoceptors are expressed in specialized smooth [...] Read more.
The G protein-coupled α2-adrenoceptor subtype C (abbreviated α2C-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 α2C-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 α2C-AR C-terminus, when introduced as competitor peptides, to selectively inhibit intracellular α2C-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 α2C-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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Figure 1

Figure 1
<p>Endogenous α<sub>2C</sub>-adrenoceptor translocation in murine microVSM. The effect of solvent DMSO (16 h, (<b>A</b>)) compared with the effect of the adenylyl cyclase activator forskolin (16 h, (<b>B</b>)) on endogenous α<sub>2C</sub>-adrenoceptor translocation when added to quiescent primary murine microVSM, determined by immunofluorescence (Alexa Fluor 568 red, α<sub>2C</sub>-adrenoceptors; DAPI blue, nucleus). A similar response is seen in human-derived microVSM [<a href="#B29-ijms-24-17558" class="html-bibr">29</a>]. The arrows in (<b>A</b>) point to intracellular receptors which are localized in the perinuclear region, whereas the arrows in (<b>B</b>) point to receptors on the cell surface (referred to as the cell boundary). Scale bar = 50 µm, 60× oil objective.</p>
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<p>Transient transfection of α<sub>2C</sub>-AR-GFP in murine microVSM. Recombinant α<sub>2C</sub>-adrenoceptor tagged with green fluorescent protein (GFP) transiently transfected in murine tail artery explanted vascular smooth muscle cells. The intracellular localization of the receptor can be seen in the perinuclear region under quiescent, unstimulated conditions, similar to endogenous α<sub>2C</sub>-adrenoceptors in <a href="#ijms-24-17558-f001" class="html-fig">Figure 1</a>A. Scale bar = 50 µm, 40× objective.</p>
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<p>Transient co-transfections in NIH/3T3 cells using HA-α<sub>2C</sub>-adrenoceptors (HA-α<sub>2C</sub>-AR). Cells were co-transfected with HA-α<sub>2C</sub>-AR along with an empty expression vector (pcDNA, (<b>A</b>)) or with constitutively active Rap1A (Rap1A-CA, (<b>B</b>)). The arrows in (<b>A</b>) point to intracellular receptors which are localized in the perinuclear region, whereas the arrows in (<b>B</b>) point to receptors on the cell surface (referred to as the cell boundary), similar to the endogenous receptors in <a href="#ijms-24-17558-f001" class="html-fig">Figure 1</a>. Scale bar = 10 µm, 60× oil objective.</p>
Full article ">Figure 4
<p>Transient co-transfections in NIH/3T3 cells using α<sub>2C</sub>-AR-GFP. Cells were co-transfected with α<sub>2C</sub>-AR-GFP along with an empty expression vector (pcDNA, (<b>A</b>)) or with constitutively active Rap1A (Rap1A-CA, (<b>B</b>)). The arrows in (<b>A)</b> point to intracellular receptors which are localized in the perinuclear region, whereas the arrows in (<b>B)</b> point to receptors on the cell surface (referred to as the cell boundary), similar to the endogenous receptors and recombinant HA-tagged receptors in <a href="#ijms-24-17558-f001" class="html-fig">Figure 1</a> and <a href="#ijms-24-17558-f003" class="html-fig">Figure 3</a>, respectively. Scale bar = 10 µm, 60× oil objective.</p>
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<p>Transient co-transfections of wild-type α<sub>2A</sub>-adrenoceptors and α<sub>2A</sub>-<sub>2C</sub>-AR chimera. Cells were co-transfected with α<sub>2A</sub>-adrenoceptor and the control expression vector pcDNA (<b>A</b>) or with constitutively active Rap1A (Rap1A-CA) (<b>B</b>). Similarly, the α<sub>2A</sub>-<sub>2C</sub>-AR chimera was co-transfected with pcDNA (<b>C</b>) or with Rap1A-CA (<b>D</b>). The receptors at the cell boundary were assessed by quantitating the mean fluorescence intensity at six random regions of interest (ROI) on the cell boundary per cell. The data from the (n) number of cells analyzed for each construct are shown; *** <span class="html-italic">p</span> &lt; 0.0001 (<b>E</b>). The arrows point to receptor localization under the tested conditions. The scale bar = 10 µm, 40× objective. The data presented are available in the <a href="#app1-ijms-24-17558" class="html-app">Supplementary Materials</a>.</p>
Full article ">Figure 6
<p>Transient co-transfections of wild-type α<sub>2C</sub>-adrenoceptor and α<sub>2C</sub>-<sub>2A</sub>-AR chimera. Cells were co-transfected with α<sub>2C</sub>-adrenoceptor and the control expression vector pcDNA (<b>A</b>), or with constitutively active Rap1A (Rap1A-CA) (<b>B</b>). Similarly, the α<sub>2C</sub>-<sub>2A</sub>-AR chimera was co-transfected with pcDNA (<b>C</b>) or with Rap1A-CA (<b>D</b>). The receptors at the cell boundary were assessed by quantitating the mean fluorescence intensity at six random regions of interest (ROI) on the cell boundary per cell. The data from the (n) number of cells analyzed for each construct are shown; *** <span class="html-italic">p</span> &lt; 0.0001 (<b>E</b>). The arrows point to receptor localization under the tested conditions. The scale bar = 10 µm, 60× oil objective. The data presented are available in the <a href="#app1-ijms-24-17558" class="html-app">Supplementary Materials</a>.</p>
Full article ">Figure 6 Cont.
<p>Transient co-transfections of wild-type α<sub>2C</sub>-adrenoceptor and α<sub>2C</sub>-<sub>2A</sub>-AR chimera. Cells were co-transfected with α<sub>2C</sub>-adrenoceptor and the control expression vector pcDNA (<b>A</b>), or with constitutively active Rap1A (Rap1A-CA) (<b>B</b>). Similarly, the α<sub>2C</sub>-<sub>2A</sub>-AR chimera was co-transfected with pcDNA (<b>C</b>) or with Rap1A-CA (<b>D</b>). The receptors at the cell boundary were assessed by quantitating the mean fluorescence intensity at six random regions of interest (ROI) on the cell boundary per cell. The data from the (n) number of cells analyzed for each construct are shown; *** <span class="html-italic">p</span> &lt; 0.0001 (<b>E</b>). The arrows point to receptor localization under the tested conditions. The scale bar = 10 µm, 60× oil objective. The data presented are available in the <a href="#app1-ijms-24-17558" class="html-app">Supplementary Materials</a>.</p>
Full article ">Figure 7
<p>Western blotting and the detection of expressed peptides. SDS-PAGE peptide separation of pTAT–HA–A2A–AR<sup>438–450</sup> and pTAT–HA–A2C–AR<sup>449–462</sup> expressed in BL21 (DE3)-STAR on 16% resolving gel. The peptides were detected using an anti-HA monoclonal antibody (clone 2-2.2.14), targeting the HA-tag on the peptides. Supernatant (sup).</p>
Full article ">Figure 8
<p>Peptide delivery and localization. The peptide staining was distinct from the background signal, which was assessed for α<sub>2C</sub>-AR-GFP/Rap1A-CA co-transfected cells without peptides and without or with primary anti-HA antibody (<b>A</b>–<b>D</b>). The cellular uptake of both peptides was observed, with localization in the cytosol and/or the perinuclear region for pTAT–HA–A2A–AR<sup>438–450</sup> (Peptide A) (<b>E</b>–<b>G</b>) and pTAT–HA–A2C–AR<sup>449–462</sup> (Peptide C) (<b>H</b>–<b>J</b>). The arrows point to α<sub>2C</sub>-AR-GFP localization. The scale bar = 10 µm, 60× oil objective.</p>
Full article ">Figure 8 Cont.
<p>Peptide delivery and localization. The peptide staining was distinct from the background signal, which was assessed for α<sub>2C</sub>-AR-GFP/Rap1A-CA co-transfected cells without peptides and without or with primary anti-HA antibody (<b>A</b>–<b>D</b>). The cellular uptake of both peptides was observed, with localization in the cytosol and/or the perinuclear region for pTAT–HA–A2A–AR<sup>438–450</sup> (Peptide A) (<b>E</b>–<b>G</b>) and pTAT–HA–A2C–AR<sup>449–462</sup> (Peptide C) (<b>H</b>–<b>J</b>). The arrows point to α<sub>2C</sub>-AR-GFP localization. The scale bar = 10 µm, 60× oil objective.</p>
Full article ">Figure 9
<p>Peptide specificity to inhibit receptor translocation. Transient co-transfection experiments were performed using an α<sub>2C</sub>-AR-GFP reporter along with the control expression vector pcDNA or Rap1A-CA in the absence or presence of pTAT–HA–A2A–AR<sup>438–450</sup> (Peptide A) or pTAT–HA–A2C–AR<sup>449–462</sup> (Peptide C). The receptors at the cell boundary were assessed by quantitating the mean fluorescence intensity at six random regions of interest (ROI) on the cell boundary per cell. The data from the (n) number of cells analyzed for each set of experiments are shown; (*** <span class="html-italic">p &lt;</span> 0.0001; ns, not significant). The data presented are available in the <a href="#app1-ijms-24-17558" class="html-app">Supplementary Materials</a>.</p>
Full article ">Figure 10
<p>Human microvascular smooth muscle cell Peptide C delivery and effect on endogenous α<sub>2C</sub>-adrenoceptors. (<b>A</b>–<b>D</b>) Delivery of HA-TAT-α<sub>2C</sub>-AR (Peptide C) to human microVSM and effect on receptor translocation examined by immunofluorescence microscopy (α<sub>2C</sub>-adrenoceptors, Alexa Fluor 568, red; Peptide C, AlexaFluor 488, green; nucleus, blue). Cells were treated with the cAMP analog and Epac-Rap1A activator 8-pCPT-2′-O-Me-cAMP (100 µM, 16 h). The arrows point to cell boundary. Scale bar = 20 µm. The α<sub>2C</sub>-adrenoceptors are perinuclear in the presence of the peptide. (<b>E</b>) Quantification of cell boundary α<sub>2C</sub>-adrenoceptors. The receptors at the cell boundary were assessed by quantitating the mean fluorescence intensity at four random regions of interest (ROI) on the cell boundary per cell. The data from the (n) number of cells analyzed for each set of experiments are shown; (*** <span class="html-italic">p &lt;</span> 0.0001). In the presence of Peptide C, the cell surface localization of the receptor was below the baseline level in unstimulated cells (red box). (<b>F</b>) Assessing microVSM receptor function by measuring intracellular levels of cAMP in 8-pCPT-2′-O-Me-cAMP (100 µM, 14 h) stimulated cells in the absence or presence of Peptide C. The data are corrected for baseline cAMP level and shown as the percent response to forskolin alone, expressed as mean ± SEM for four independent replicates (see <a href="#sec4-ijms-24-17558" class="html-sec">Section 4</a> for details of the assay; * <span class="html-italic">p</span> &lt; 0.05). The α<sub>2</sub>-adrenoceptors are Gi-coupled, and activation by the agonist UK, 14,304 inhibits adenylyl cyclase and intracellular cAMP levels. The data presented are available in the <a href="#app1-ijms-24-17558" class="html-app">Supplementary Materials</a>.</p>
Full article ">Figure 10 Cont.
<p>Human microvascular smooth muscle cell Peptide C delivery and effect on endogenous α<sub>2C</sub>-adrenoceptors. (<b>A</b>–<b>D</b>) Delivery of HA-TAT-α<sub>2C</sub>-AR (Peptide C) to human microVSM and effect on receptor translocation examined by immunofluorescence microscopy (α<sub>2C</sub>-adrenoceptors, Alexa Fluor 568, red; Peptide C, AlexaFluor 488, green; nucleus, blue). Cells were treated with the cAMP analog and Epac-Rap1A activator 8-pCPT-2′-O-Me-cAMP (100 µM, 16 h). The arrows point to cell boundary. Scale bar = 20 µm. The α<sub>2C</sub>-adrenoceptors are perinuclear in the presence of the peptide. (<b>E</b>) Quantification of cell boundary α<sub>2C</sub>-adrenoceptors. The receptors at the cell boundary were assessed by quantitating the mean fluorescence intensity at four random regions of interest (ROI) on the cell boundary per cell. The data from the (n) number of cells analyzed for each set of experiments are shown; (*** <span class="html-italic">p &lt;</span> 0.0001). In the presence of Peptide C, the cell surface localization of the receptor was below the baseline level in unstimulated cells (red box). (<b>F</b>) Assessing microVSM receptor function by measuring intracellular levels of cAMP in 8-pCPT-2′-O-Me-cAMP (100 µM, 14 h) stimulated cells in the absence or presence of Peptide C. The data are corrected for baseline cAMP level and shown as the percent response to forskolin alone, expressed as mean ± SEM for four independent replicates (see <a href="#sec4-ijms-24-17558" class="html-sec">Section 4</a> for details of the assay; * <span class="html-italic">p</span> &lt; 0.05). The α<sub>2</sub>-adrenoceptors are Gi-coupled, and activation by the agonist UK, 14,304 inhibits adenylyl cyclase and intracellular cAMP levels. The data presented are available in the <a href="#app1-ijms-24-17558" class="html-app">Supplementary Materials</a>.</p>
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<p>Impact of increased intracellular α<sub>2</sub>-adrenoceptors on VSM constriction at warm and cold temperatures. In the early stage, vascular stress (for example, injury due to cycles of vasospastic attacks or mechanical, followed by inflammation) elevates cyclooxygenase-2 (COX-2), intracellular cyclic AMP (cAMP), transcriptionally increasing α<sub>2C</sub>-adrenoceptors and the intracellular pool of receptors (warm). Cooling mobilizes α<sub>2C</sub>-adrenoceptors to the cell surface, causing vasoconstriction (cold). Vascular injury contributes to the impaired dilator (nitric oxide (NO)—cyclic guanosine monophosphate, cGMP) function of endothelium and increases vasoconstriction via α<sub>2C</sub>-adrenoceptors during the disease process. The cyclic AMP-Rap1A signaling predominates. There is, therefore, a loss of balance between vasodilation and vasoconstriction, tipping in favor of vasoconstriction. Individuals with the variant α<sub>2A</sub>-adrenoceptor (α<sub>2A</sub>-AR) rs7090046 and NOS3 variant rs3918226 have an increased expression of α<sub>2A</sub>-adrenoceptors and reduced vasodilator NO, contributing to a severe clinical condition versus the mild condition in individuals without these variants. The colors denote α<sub>2A</sub>-adrenoceptors (<span style="color:#00B050">2A, green</span>), and α<sub>2C</sub>-adrenoceptors (<span style="color:red">2C, red</span>).</p>
Full article ">Figure 12
<p>Plasmid constructs for domain-swapping studies. (<b>A</b>) Full-length amino terminus HA-tagged wild-type receptor α<sub>2A</sub>- <span style="color:#00B050">(green)</span> and α<sub>2C</sub>-adrenoceptor <span style="color:red">(red) </span>and (<b>B</b>) chimeras generated for the studies. Carboxyl-termini and part of transmembrane 7 (TMVII) are shown. Putative regulatory regions are indicated, including NPXXY, FXXXFXXXF, and a non-conserved α<sub>2C</sub>-adrenoceptor arginine-rich region (R-454–458). Single letter amino acid codes are shown.</p>
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<p>DNA fragments with restriction enzyme overhangs harboring α<sub>2</sub>-adrenoceptor subtypes A and C regions of interest. These fragments were used to generate DNA constructs pTAT–HA–A2A–AR<sup>438–450</sup> (WT) and pTAT–HA–A2C–AR<sup>449–462</sup> (WT) for peptide bacterial expression.</p>
Full article ">Figure 14
<p>The amino acid sequence of the peptides used in the study. The peptides, including (<b>A</b>) pTAT–HA–A2A–AR<sup>438–450</sup> (WT) and (<b>B</b>) pTAT–HA–A2C–AR<sup>449–462</sup> (WT), were expressed using the bacterial expression vector pTAT-HA. The peptides include the in-frame fusion of 6X-Histidine (HHHHHH) for nickel column purification, TAT protein transduction domain (YGRKKRRQRRR) for cellular delivery, and hemagglutinin (HA, YPYDVPDYA) tag for the detection of peptides by anti-HA antibody in Western blotting and immunofluorescence microscopy.</p>
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20 pages, 2516 KiB  
Article
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 2170
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
<p>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 (<b>A</b>) TXNDC5 and (<b>B</b>) HSPA9. (<b>C</b>) AML12 WT and KO were co-immunoprecipitated with the PRDX6 antibody, and (<b>D</b>) the precipitates were examined via Western blotting with the anti-TXNDC5 antibody. Description of the lanes from left to right (<b>A</b>–<b>C</b>): 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. (<b>D</b>) 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.</p>
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<p>(<b>A</b>,<b>B</b>) Expression pattern of HSPA9. mRNA level of <span class="html-italic">Hspa9</span> in the (<b>A</b>) AML12 cell line. (<b>B</b>) Protein level of HSPA9 and its Western blot in the hepatic cell line. (<b>C</b>–<b>E</b>) Expression pattern of PRDX6. The RNA expression level of <span class="html-italic">Prdx6</span> (<b>C</b>) in the AML12 cell line and (<b>D</b>) the mRNA level of <span class="html-italic">Prdx6b</span> is shown in the AML12 cell line. (<b>E</b>) 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; * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. (<b>F</b>) 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.)</p>
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<p>(<b>A</b>) Lipid peroxidation analysis. Significant MDA reduction in the KO cells is shown. (<b>B</b>–<b>D</b>) Assessment of glutathione and iPLA<sub>2</sub> activities. Considerable decrements of (<b>B</b>) GSH and (<b>C</b>) iPLA<sub>2</sub> activity in the KO cells are indicated. (<b>D</b>) AML12 KO cells display a remarkable increment in the total PLA<sub>2</sub>. Statistical analysis was carried out according to the Mann–Whitney U test for pairwise comparisons; * <span class="html-italic">p</span> &lt; 0.05, *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>(<b>A</b>–<b>C</b>) Possible protein–protein interactions of mouse (<b>A</b>) TXNDC5, (<b>B</b>) PRDX6 and (<b>C</b>) 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. (<b>D</b>) 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 (SL<sub>2</sub>R &gt; ±2 in the <span class="html-italic">Prdx6</span> dataset and SL<sub>2</sub>R &gt; ±1 in the <span class="html-italic">Hspa9</span> dataset). The liver protein abundance (parts per million (ppm)) based on the PAX database was considered as a final step (PPM &gt; 300 in the <span class="html-italic">Prdx6</span> dataset and PPM &gt; 100 in the <span class="html-italic">Hspa9</span> dataset). Expression of (<b>E</b>) <span class="html-italic">Hspa9-</span> and (<b>F</b>) <span class="html-italic">Prdx6</span>-associated genes in the AML12 cell line. Data are mean ± SD. Statistical analysis was performed according to the Mann–Whitney U test. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01.</p>
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<p>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.</p>
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Review

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15 pages, 304 KiB  
Review
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 2438
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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