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Protein Structure and Cancer
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Protein Structure and Cancer

A special issue of Cancers (ISSN 2072-6694).

Deadline for manuscript submissions: closed (30 April 2023) | Viewed by 21671

Special Issue Editor

Dr. Michalis Aivaliotis

E-Mail Website
Guest Editor
Laboratory of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, 54636 Thessaloniki, Greece
Interests: functional proteomics; cancer biology; neurodegenerative diseases; system biology; bioinformatics
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Proteins are fundamental molecules to all living organisms and the remarkable way they fold into complex three-dimensional (3D) structures strongly defines organism’s functional capabilities and phenotype. Protein structure is highly dynamic and defines protein function and interactions. The dynamic feature of protein structure enhances and supports the amazing flexibility of cellular and extracellular biochemistry and cells’ inherent capability for autoregulation and adaptation to different conditions. On the other hand, a problematic protein structure may lead to protein deregulation, dysfunction, or no function, and consequently to disease or even death. Protein structural alterations are a hallmark of several diseases, including cancer. In addition, such alterations in cancer lead to diverse protein functions that can significantly promote cancerous transformation and phenotype. Local concentrations of mutations are well known in human cancers affecting protein function and interactions enhancing cancer development and progression, contributing to tumor heterogeneity and tumorigenesis. However, their 3D spatial relationships in the encoded proteins have yet to be systematically investigated. It is expected that the elucidation of protein structural alterations resulting from such driver mutations is of paramount importance for obtaining an in-depth understanding of the molecular mechanisms leading to cancer. Such an understanding can provide more direct and clinically relevant knowledge and evidence of disease states than genetic signatures alone.

We are excited to invite authors to submit original research and review articles that address the progress and current standing of protein-structure-based studies in cancer research. Research areas may include various aspects and approaches that have been developed for understanding and predicting the role of protein structure alterations in the development, progression, and resistance to therapy of different cancers, as well as their potential application in developing precision cancer therapies.

Prof. Dr. Michalis Aivaliotis
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cancers is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • protein structure dynamics
  • structural cancer biology
  • protein pathways in cancer
  • structure–function alterations
  • in silico protein structure prediction
  • protein interactions
  • cancer mutations and protein structure alterations

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Published Papers (7 papers)

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27 pages, 18527 KiB  
Article
Interruption of p53-MDM2 Interaction by Nutlin-3a in Human Lymphoma Cell Models Initiates a Cell-Dependent Global Effect on Transcriptome and Proteome Level
by Konstantina Psatha, Laxmikanth Kollipara, Elias Drakos, Elena Deligianni, Konstantinos Brintakis, Eustratios Patsouris, Albert Sickmann, George Z. Rassidakis and Michalis Aivaliotis
Cancers 2023, 15(15), 3903; https://doi.org/10.3390/cancers15153903 - 31 Jul 2023
Cited by 7 | Viewed by 2325
Abstract
In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 [...] Read more.
In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
►▼ Show Figures

Figure 1

Figure 1
<p>Overview of the results from the comparative transcriptomics and proteomics analysis on model lymphoma cell lines +/−N3a. (<b>A</b>): Venn diagrams of the overlapping deregulated mRNAs and deregulated proteins demonstrate the cell-specific effect of N3a on the lymphoma proteome and transcriptome. Bar diagrams show the exact number of proteins deregulated (in blue), up-regulated (in red) and down-regulated (in green) in cHL and NHLs. (<b>B</b>): The acquired data were hierarchically clustered, displaying in heat-maps the differential expression of mRNAs/proteins +/−N3a (FDR-adjusted, <span class="html-italic">p</span>-value &lt; 0.05). Functional annotation analysis was combined with mRNA/protein expression level data to identify the top-GO-term enrichment categories (BP, CC and MF) relevant to N3a’s effect in both omics analyses. Columns were clustered according to top-ranked-categories of interest, and rows were clustered according to the lymphoma-subtype. Presence and enrichment levels were color-coded in a red-black gradient representing the −log10 (adjusted <span class="html-italic">p</span>-value) (red: presence in treated vs. control; black: missing value), with intensity proportional to presence/enrichment. (<b>C</b>): Selected pathways with a significant enrichment in proteins affected by N3a treatment indicating an up- or down-regulation of these pathways.</p> Full article ">Figure 2
<p>Common deregulated proteins and mRNAs in three lymphoma types: Heatmap and bar plot image depicting a common pattern of changes in mRNA and protein expression for the 12 genes that were commonly deregulated in both omics analyses, corresponding to the three lymphoma cell lines of our study (FDR-adjusted, <span class="html-italic">p</span>-value &lt; 0.05). Protein interaction network of the common differentially expressed proteins using STRING is also shown.</p> Full article ">Figure 3
<p>Protein interaction network of deregulated global common proteome profile of N3a-treated cHL and NHL lymphomas: Visualization of the common differentially regulated proteins (FDR-adjusted, <span class="html-italic">p</span>-value &lt; 0.05) in cHL/NHL in a protein–protein interaction network using STRING and Cytoscape. Nodes indicate proteins and edges indicate the interactions between them. The up- (red) or down-regulation (green) is color coded for each protein (both colors: differential protein-regulation in the three lymphomas). Unsupervised clustering of lymphoma-subtype proteome alterations clustered the deregulated proteins into related functional classes, including down-regulated cell cycle, RNA splicing, ribosome, chromatin remodeling/modification apoptosis, proteolysis, metabolic processes, DNA repair, ribosome, and oxidative phosphorylation. Bar diagrams in the right bottom area of the figure represent the percentage of proteins commonly deregulated (up-regulated: red; down-regulated: green) or differentially deregulated (grey) in cHL/NHLs.</p> Full article ">Figure 4
<p>Integrative visualization of the N3a effect in p53 signaling pathway: Venn diagrams in the upper left half of the figure illustrate the number of the: (1) molecules in both omics datasets, (2) deregulated proteins and (3) deregulated mRNAs, emphasizing on the divergent effect N3a has on the lymphoma proteome and transcriptome. Color-code bar-plot images in the upper right half of the figure (red: up-regulation; green: down-regulation) depict an overall common pattern of differential expression at the mRNA/protein level for the deregulated p53-related-genes in both omics analyses, corresponding to the three lymphoma cell lines of our study. Integrative protein–protein interaction network of the N3a-effect on p53-related-genes is visualized in the center of the figure using STRING and Cytoscape. Nodes indicate proteins and edges indicate the interactions between them, while color depicts regulation (red: increase; green: decrease). Around the networks, representative p53-selected proteins clustered according to their involvement to other pathways are shown in green–red–black gradient, representing the −log10 (adjusted <span class="html-italic">p</span>-value) (red: higher abundance in treated; green: lower abundance in treated; black: missing value). Color intensity reflects differences in abundance levels. The western blot image of TP3 and MDM2 is also included with the bar graphs showing the ratio of each band after the densitometry analysis.</p> Full article ">Figure 5
<p>Regulation profile of key cellular pathways and biological processes by N3a in the three lymphoma types: The scheme summarizes N3a-mediated alterations in eight core cellular pathways in cHL/NHL cells. Each panel demonstrates the regulation profile of a cellular pathway post-N3a-treatment. Venn diagrams are generated as in <a href="#cancers-15-03903-f004" class="html-fig">Figure 4</a>. Bar-plot images in the right half of each panel demonstrate the differential regulation status of mRNAs/proteins relevant to every cellular pathway, in each lymphoma cell line of our study (red: up-regulation; green: down-regulation).</p> Full article ">Figure 6
<p>mTOR- and PI3K/AKT-related protein expression levels in N3a-treated cHL, MCL and ALK+ ALCL cells: (<b>A</b>): Venn diagrams and color-coded bar-plot images are generated as in <a href="#cancers-15-03903-f004" class="html-fig">Figure 4</a>. Bar-plot images highlight the differential mRNA/protein levels for the mTOR-related- and PI3K/AKT-related- genes that are deregulated in both omics analyses, in each lymphoma cell line of our study (red: up-regulation; green: down-regulation). (<b>B</b>): Key mTOR pathway-proteins showed decreased or non-regulated expression levels. Proteomic results were validated using Western blot in cHL, MCL, and ALK+ ALCL cells +/−N3a. Stabilization and activation of wt p53 suppressed mTOR signaling in HL, MCL, and ALK+ ALCL cells. β-Actin protein expression was used as loading control. The calculated intensity and ratio of each band after the densitometry analysis are available in <a href="#app1-cancers-15-03903" class="html-app">Table S13</a>.</p> Full article ">Figure 7
<p>Heat shock proteins expression levels in N3a-treated HL, MCL and ALK + ALCL cells: (<b>A</b>): Venn diagrams and color-coded bar-plot images are generated as in <a href="#cancers-15-03903-f004" class="html-fig">Figure 4</a>. Bar-plot displayed primarily a down-regulation of the HSPs-related-genes following N3a on the protein level in a lymphoma-cell-specific manner (red: up-regulation; green: down-regulation). (<b>B</b>): HSP70 (nine family members) and HSP90 (three family members) were found to have decreased protein levels by omics (right panel) and were validated using WB analysis. β-Actin protein expression served as a protein load and integrity control. The bar graphs showing the ratio of each band after the densitometry analysis are also included in this figure.</p> Full article ">Figure 8
<p>Synergistic effect of N3a with HSPs and mTOR inhibitors in lymphoma cells: Summary of the therapeutic scheme followed in our study, with major findings highlighted in bold. Color-coded arrows represent regulation (red: up-, green: down-). The signaling pathways in which PI3K, mTOR, MDM2, p53, and Hsp90 are participating, are in a continue balance of inter- and intra-activation and deactivation towards cellular homeostasis and fitness. Every imbalance in their regulation supports lymphomagenesis, therefore are targets of promising therapeutic strategies. In the present study we use synergistically four inhibitors of these proteins, namely LY294002, Rapa, N3a, 17-AGG, respectively, aiming to suppress lymphoma cell growth.</p> Full article ">
21 pages, 3455 KiB  
Article
Mutation in the Common Docking Domain Affects MAP Kinase ERK2 Catalysis and Stability
by Leonore Novak, Maria Petrosino, Alessandra Pasquo, Apirat Chaikuad, Roberta Chiaraluce, Stefan Knapp and Valerio Consalvi
Cancers 2023, 15(11), 2938; https://doi.org/10.3390/cancers15112938 - 26 May 2023
Cited by 3 | Viewed by 1828
Abstract
The extracellular-signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK) located downstream of the Ras-Raf-MEK-ERK signal transduction cascade, is involved in the regulation of a large variety of cellular processes. The ERK2, activated by phosphorylation, is the principal effector of a central signaling [...] Read more.
The extracellular-signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK) located downstream of the Ras-Raf-MEK-ERK signal transduction cascade, is involved in the regulation of a large variety of cellular processes. The ERK2, activated by phosphorylation, is the principal effector of a central signaling cascade that converts extracellular stimuli into cells. Deregulation of the ERK2 signaling pathway is related to many human diseases, including cancer. This study reports a comprehensive biophysical analysis of structural, function, and stability data of pure, recombinant human non-phosphorylated (NP-) and phosphorylated (P-) ERK2 wild-type and missense variants in the common docking site (CD-site) found in cancer tissues. Because the CD-site is involved in interaction with protein substrates and regulators, a biophysical characterization of missense variants adds information about the impact of point mutations on the ERK2 structure–function relationship. Most of the P-ERK2 variants in the CD-site display a reduced catalytic efficiency, and for the P-ERK2 D321E, D321N, D321V and E322K, changes in thermodynamic stability are observed. The thermal stability of NP-ERK2 and P-ERK2 D321E, D321G, and E322K is decreased with respect to the wild-type. In general, a single residue mutation in the CD-site may lead to structural local changes that reflects in alterations in the global ERK2 stability and catalysis. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
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Figure 1

Figure 1
<p>The ERK1/2 cascade and ERK2 structure. (<b>A</b>) ERK1/2 signaling cascade. The ERK kinases from the cytoplasm can be translocated into the nucleus and catalyze the phosphorylation of downstream targets upon activation. Mitogen-activated protein kinase 1 and 2 (MEK1 and MEK2) activate the ERK2 by phosphorylation of two residues, Thr185 and Tyr187. (<b>B</b>) Human ERK2 structure in complex with an inhibitor (pdb: 4zzn) [<a href="#B12-cancers-15-02938" class="html-bibr">12</a>]. Thr185 and Tyr187 residues, involved in the activation of ERK2, are evidenced in sticks; the inhibitor is not shown.</p> Full article ">Figure 2
<p>Model of the human non-phosphorylated-ERK2 (NP-ERK2) CD-site. The CD-site is the common docking site that contains residues that interact with protein substrates and is composed of two negatively charged residues, D318 and D321, depicted in sticks. In orange, the docking peptide derived from hematopoietic tyrosine phosphatase, a negative regulator of ERK2, sequence in a one letter code: RLQERRGSNVALMLDC. In the NP-ERK2 structure (pdb: 2gph), the docking peptide is involved in extensive electrostatic interactions with the CD-site. All the other relevant parts of the ERK2 structure are shown: in dark blue and in light green, the activation loop and the glycine-rich loop, respectively; in purple and in light blue, the helices alphaC and C-terminal, respectively; in the grey circle, the F-site. The T185 and Y187 at the active site are represented in sticks.</p> Full article ">Figure 3
<p>Spectral properties of the non-phosphorylated-ERK2 (NP-ERK2) and phosphorilated-ERK2 (P-ERK2) wild-types. (<b>A</b>) Near-UV CD spectra were recorded in a 1.0-cm quartz cuvette at 1.3 mg/mL protein concentration in 20 mM Tris-HCl, pH 7.5, containing 1.0 mM DTT and 0.1 M NaCl. (<b>B</b>) Intrinsic fluorescence emission spectra (295 nm excitation wavelength) were monitored at 130 µg/mL (0.08 AU 280 nm) in 20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 0.2 mM DTT. (<b>C</b>) Far-UV CD spectra were monitored in a 0.1-cm quartz cuvette at 130–170 µg/mL in 20 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and 0.2 mM DTT. The continuous lines are used for the NP-ERK2, the dashed lines are used for the P-ERK2. All spectra were recorded at 20 °C.</p> Full article ">Figure 4
<p>Spectral properties of the non-phosphorylated-ERK2 (NP-ERK2) and phosphorylated-ERK2 (P-ERK2) variants in the CD-site. Near-UV CD spectra of the NP-ERK2, continuous line (<b>A</b>), and of P-ERK2 variants, medium-dashed line (<b>B</b>), were monitored at 1.3 mg/mL protein concentration in 20 mM Tris-HCl pH 7.5, 1.0 mM DTT, and 0.1M NaCl, in a 1.0-cm quartz cuvette. Intrinsic fluorescence emission spectra of the NP-ERK2, continuous line (<b>C</b>), and of the P-ERK2 variants, dashed lines (<b>D</b>), were recorded at 130 μg/mL (0.08 AU 280 nm, 295 nm excitation wavelength), in 20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 0.2 mM DTT.</p> Full article ">Figure 5
<p>Thermal unfolding of the non-phosphorylated-ERK2 (NP-wild-type) and phosphorylated-ERK2 (P-wild-type) wild-types. The ERK2 wild-type proteins (100–130 μg/mL), in 20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, and 200 µM DTT, were heated from 20 °C to 90 °C. The molar ellipticity at 222 nm ([Θ]<sub>222</sub>) was monitored continuously every 0.5 °C and normalized. The first derivative of the thermal transition data is shown in the inset.</p> Full article ">Figure 6
<p>Effect of the binding with KIM peptide of DUSP6 on the amount of heat released or absorbed during association of the phosphorylated-ERK2 (P-ERK2) wild-type (<b>A</b>), and the P-ERK2 D321N variant (<b>B</b>). The measurements were performed using an “Affinity ITC” (TA-Instrument) with 500µM of peptide (KIM of DUSP6) in the syringe, and 93 µM of the P-ERK2 wild-type and 113 µM of the P-ERK2 D321N in the cell at 30 °C. The K<sub><span class="html-small-caps">D</span></sub> was 14.4 µΜ for the phosphorylated wild-type (<b>A</b>), no binding was measured for the phosphorylated D321N (<b>B</b>).</p> Full article ">Figure 7
<p>The human phosphorylated-ERK2 (P-ERK2) D321N (in red) bound to inhibitor AZD0364 (in cyan). In green, the activation loop that begins with the sequence DFG and ends with the sequence APE. The dotted line underlines the undefined region of the three TEY amino acids, important for the activation of ERK2. In cyan, the compound AZD0364 that binds in two opposite regions the mutant D321N.</p> Full article ">Figure 8
<p>Structural comparison of the human phosphorylated-ERK2 (P-ERK2) D321N bound to inhibitor AZD0364 (in cyan). Superposition of the human P-ERK2 D321N (in red), rat P-ERK2 wild-type (in yellow, pdb: 2erk), and rat NP-ERK2 D321N (in gray, pdb: 6ot6). The residues T185 and Y187, important for the activation of ERK2, are depicted in sticks. The dotted line underlines the undefined region of the three TEY amino acids involved in ERK2 activation.</p> Full article ">
22 pages, 20801 KiB  
Article
E2 Partner Tunes the Ubiquitylation Specificity of Arkadia E3 Ubiquitin Ligase
by Georgia N. Delegkou, Maria Birkou, Nefeli Fragkaki, Tamara Toro, Konstantinos D. Marousis, Vasso Episkopou and Georgios A. Spyroulias
Cancers 2023, 15(4), 1040; https://doi.org/10.3390/cancers15041040 - 7 Feb 2023
Viewed by 2385
Abstract
Arkadia (RNF111) is a positive regulator of the TGF-β signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions [...] Read more.
Arkadia (RNF111) is a positive regulator of the TGF-β signaling that mediates the proteasome-dependent degradation of negative factors of the pathway. It is classified as an E3 ubiquitin ligase and a SUMO-targeted ubiquitin ligase (STUBL), implicated in various pathological conditions including cancer and fibrosis. The enzymatic (ligase) activity of Arkadia is located at its C-terminus and involves the RING domain. Notably, E3 ligases require E2 enzymes to perform ubiquitylation. However, little is known about the cooperation of Arkadia with various E2 enzymes and the type of ubiquitylation that they mediate. In the present work, we study the interaction of Arkadia with the E2 partners UbcH5B and UbcH13, as well as UbcH7. Through NMR spectroscopy, we found that the E2–Arkadia interaction surface is similar in all pairs examined. Nonetheless, the requirements and factors that determine an enzymatically active E2–Arkadia complex differ in each case. Furthermore, we revealed that the cooperation of Arkadia with different E2s results in either monoubiquitylation or polyubiquitin chain formation via K63, K48, or K11 linkages, which can determine the fate of the substrate and lead to distinct biological outcomes. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
►▼ Show Figures

Figure 1

Figure 1
<p>Sequence alignment of the UBC domain of different E2 enzymes produced by Jalview. Residues are colored according to the percentage of the residues in each column that agree with the sequence of UbcH5B (&gt;80% dark blue, &gt;60% medium dark blue, &gt;40% light blue, ≤40% no color). The conserved phenylalanine and SPA motif at the L1 and L2 loops, respectively, are highlighted with red boxes.</p> Full article ">Figure 2
<p>Interaction of E3 Arkadia with the E2 UbcH5B F62A mutant. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio (saturation point) for <sup>15</sup>N Ark RING/<sup>14</sup>N UbcH5B F62A, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. (<b>b</b>) Ark RING mapping (PDBid: 2KIZ) after addition of UbcH5B F62A (residues that disappeared during the interaction are colored red, whereas residues that exhibited ‘fast exchange’ interactions are colored coral). (<b>c</b>) Diagram of the total CSPs measured at a 1:2 molar ratio for <sup>15</sup>N UbcH5B F62A/<sup>14</sup>N Ark RING. (<b>d</b>) Diagram of the total CSPs measured at a 1:2 M ratio for <sup>15</sup>N UbcH5B F62A/<sup>14</sup>N Ark LONG. (<b>e</b>) ITC data for the titration of UbcH5B F62A into Ark RING and (<b>f</b>) into Ark LONG. (<b>g</b>) In vitro auto-ubiquitylation assays of Ark LONG using UbcH5B F62A (<b>right</b>) and wt UbcH5B (<b>left</b>).</p> Full article ">Figure 3
<p>Interaction of E3 Arkadia with wt UbcH7 and UbcH7 K96S mutant. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio (saturation point) for <sup>15</sup>N Ark RING/<sup>14</sup>N wt UbcH7, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. (<b>b</b>) Ark RING mapping (PDBid: 2KIZ) after the addition of UbcH7 (residues that disappeared during the interaction are colored red, whereas residues that exhibited ‘fast exchange’ interaction are colored coral). (<b>c</b>) Diagram of the total CSPs measured at a 1:1.5 molar ratio for <sup>15</sup>N UbcH7/<sup>14</sup>N Ark RING. (<b>d</b>) UbcH7 mapping (PDBid: 6XXU) after the addition of Ark RING. (<b>e</b>) Diagram of the total CSPs measured at a 1:1.5 molar ratio for <sup>15</sup>N UbcH7 K96S/<sup>14</sup>N Ark RING. (<b>f</b>) ITC data for the titration of wt UbcH7 into Ark RING and (<b>g</b>) UbcH7 K96S into Ark RING. (<b>h</b>) In vitro auto-ubiquitylation assays of Ark LONG using wt UbcH7 and UbcH7 K96S, respectively.</p> Full article ">Figure 3 Cont.
<p>Interaction of E3 Arkadia with wt UbcH7 and UbcH7 K96S mutant. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio (saturation point) for <sup>15</sup>N Ark RING/<sup>14</sup>N wt UbcH7, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. (<b>b</b>) Ark RING mapping (PDBid: 2KIZ) after the addition of UbcH7 (residues that disappeared during the interaction are colored red, whereas residues that exhibited ‘fast exchange’ interaction are colored coral). (<b>c</b>) Diagram of the total CSPs measured at a 1:1.5 molar ratio for <sup>15</sup>N UbcH7/<sup>14</sup>N Ark RING. (<b>d</b>) UbcH7 mapping (PDBid: 6XXU) after the addition of Ark RING. (<b>e</b>) Diagram of the total CSPs measured at a 1:1.5 molar ratio for <sup>15</sup>N UbcH7 K96S/<sup>14</sup>N Ark RING. (<b>f</b>) ITC data for the titration of wt UbcH7 into Ark RING and (<b>g</b>) UbcH7 K96S into Ark RING. (<b>h</b>) In vitro auto-ubiquitylation assays of Ark LONG using wt UbcH7 and UbcH7 K96S, respectively.</p> Full article ">Figure 4
<p>Implication of the Ub<sup>B</sup> binding in Arkadia-mediated ubiquitylation. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio for <sup>15</sup>N UbcH5B S22R/<sup>14</sup>N Ark RING, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. (<b>b</b>) Diagram of the total CSPs measured at q 1:2 molar ratio for <sup>15</sup>N Ark RING/<sup>14</sup>N UbcH5B S22R. (<b>c</b>) Ark RING mapping (PDBid: 2KIZ) after the addition of UbcH5B S22R (residues that disappeared during the interaction are colored red, whereas residues that exhibited ‘fast exchange’ interaction are colored coral). (<b>d</b>) Diagram of the total CSPs measured at a 1:2.75 molar ratio for <sup>15</sup>N UbcH5B S22R/<sup>14</sup>N Ub. (<b>e</b>) Diagram of the total CSPs measured at a 1:2.75 molar ratio for <sup>15</sup>N wt UbcH5B/<sup>14</sup>N Ub. (<b>f</b>) UbcH5B mapping (PDBid: 2ESK) after the addition of Ub. (<b>g</b>) Diagram of the total CSPs measured at a 1:2.25 molar ratio for <sup>15</sup>N Ub/<sup>14</sup>N UbcH5B S22R. (<b>h</b>) Diagram of the total CSPs measured at a 1:2.25 molar ratio for <sup>15</sup>N Ub/<sup>14</sup>N wt UbcH5B. (<b>i</b>) Ub mapping (PDBid: 1D3Z) after the addition of wt UbcH5B. (<b>j</b>) In vitro auto-ubiquitylation assays of Ark LONG using UbcH5B S22R (<b>left</b>) and wt UbcH5B (<b>right</b>). (<b>k</b>) (<b>Top</b>), oxyester hydrolysis assays showing the disappearance of C85S UbcH5B-Ub and C85S S22R UbcH5B-Ub in the presence of Ark LONG, over time. (<b>Bottom</b>), densitometry quantification from gels (bars indicate the range of experimental duplicates, * indicates statistically significant difference, <span class="html-italic">p</span> value ≤ 0.05). The uncropped WB images can be seen in <a href="#app1-cancers-15-01040" class="html-app">File S1</a>.</p> Full article ">Figure 4 Cont.
<p>Implication of the Ub<sup>B</sup> binding in Arkadia-mediated ubiquitylation. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio for <sup>15</sup>N UbcH5B S22R/<sup>14</sup>N Ark RING, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. (<b>b</b>) Diagram of the total CSPs measured at q 1:2 molar ratio for <sup>15</sup>N Ark RING/<sup>14</sup>N UbcH5B S22R. (<b>c</b>) Ark RING mapping (PDBid: 2KIZ) after the addition of UbcH5B S22R (residues that disappeared during the interaction are colored red, whereas residues that exhibited ‘fast exchange’ interaction are colored coral). (<b>d</b>) Diagram of the total CSPs measured at a 1:2.75 molar ratio for <sup>15</sup>N UbcH5B S22R/<sup>14</sup>N Ub. (<b>e</b>) Diagram of the total CSPs measured at a 1:2.75 molar ratio for <sup>15</sup>N wt UbcH5B/<sup>14</sup>N Ub. (<b>f</b>) UbcH5B mapping (PDBid: 2ESK) after the addition of Ub. (<b>g</b>) Diagram of the total CSPs measured at a 1:2.25 molar ratio for <sup>15</sup>N Ub/<sup>14</sup>N UbcH5B S22R. (<b>h</b>) Diagram of the total CSPs measured at a 1:2.25 molar ratio for <sup>15</sup>N Ub/<sup>14</sup>N wt UbcH5B. (<b>i</b>) Ub mapping (PDBid: 1D3Z) after the addition of wt UbcH5B. (<b>j</b>) In vitro auto-ubiquitylation assays of Ark LONG using UbcH5B S22R (<b>left</b>) and wt UbcH5B (<b>right</b>). (<b>k</b>) (<b>Top</b>), oxyester hydrolysis assays showing the disappearance of C85S UbcH5B-Ub and C85S S22R UbcH5B-Ub in the presence of Ark LONG, over time. (<b>Bottom</b>), densitometry quantification from gels (bars indicate the range of experimental duplicates, * indicates statistically significant difference, <span class="html-italic">p</span> value ≤ 0.05). The uncropped WB images can be seen in <a href="#app1-cancers-15-01040" class="html-app">File S1</a>.</p> Full article ">Figure 5
<p>The role of R983 in Arkadia-mediated ubiquitylation. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio for <sup>15</sup>N UbcH5B/<sup>14</sup>N Ark RING R983A, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. Diagram of the total CSPs measured at a 1:2 molar ratio for (<b>b</b>) <sup>15</sup>N Ark RING R983A/<sup>14</sup>N UbcH5B, (<b>c</b>) <sup>15</sup>N UbcH5B/<sup>14</sup>N Ark RING R983K, and (<b>d</b>) <sup>15</sup>N Ark RING R983K/<sup>14</sup>N UbcH5B. (<b>e</b>) (<b>Top</b>), in vitro auto-ubiquitylation assays of Ark LONG R983A and (<b>bottom</b>), in vitro ubiquitylation assays of Ark LONG R983K. (<b>f</b>) (<b>Left</b>), oxyester hydrolysis assays showing the disappearance of C85S UbcH5B-Ub in the presence of Ark LONG, Ark LONG R983A, and Ark LONG R983K over time. (<b>Right</b>), densitometry quantification from gels (bars indicate the range of experimental duplicates, * indicates statistically significant difference, <span class="html-italic">p</span> value ≤ 0.05). (<b>g</b>) Control reaction demonstrating the stability of oxyester complex over time. The uncropped WB images can be seen in <a href="#app1-cancers-15-01040" class="html-app">File S1</a>.</p> Full article ">Figure 6
<p>Interaction of E3 Arkadia with UbcH13 E2 enzyme. (<b>a</b>) Diagram of the total CSPs measured at a 1:2 molar ratio (saturation point) for <sup>15</sup>N Ark RING/<sup>14</sup>N UbcH13, <span style="color:red">*</span>: represents disappeared residues, +: represents residues with no information. (<b>b</b>) Ark RING mapping (PDBid: 2KIZ) after the addition of UbcH13 (residues that disappeared during the interaction are colored red, whereas residues that exhibited ‘fast exchange’ interaction are colored coral). (<b>c</b>) Diagram of the total CSPs measured at a 1:2 molar ratio for <sup>15</sup>N UbcH13/<sup>14</sup>N Ark RING. (<b>d</b>) UbcH13 mapping (PDBid: 1J7D) after the addition of Ark RING. (<b>e</b>) Diagram of the total CSPs measured at a 1:2 molar ratio for <sup>15</sup>N UbcH13/<sup>14</sup>N Ark LONG. (<b>f</b>) UbcH13 mapping (PDBid: 1J7D) after the addition of Ark RING. (<b>g</b>) ITC data for the titration of UbcH13 into Ark RING and (<b>h</b>) into Ark LONG. (<b>i</b>) In vitro auto-ubiquitylation assays using UbcH13 in the presence and absence of Ark LONG (<b>left</b>) and in the presence of GST-Ark LONG or GST-Ark RING (<b>right</b>).</p> Full article ">Figure 7
<p>(<b>a</b>) In vitro ubiquitylation assays using UbcH13/MMS2 complex in the presence and absence of Ark LONG. (<b>b</b>) In vitro ubiquitylation assays using UbcH13/MMS2 complex in the presence of Ark LONG R983A or R983K.</p> Full article ">Figure 8
<p>In vitro ubiquitylation assays using UbcH5B in the presence of (<b>a</b>) Ub K48R and wt Ub, (<b>b</b>) Ub K11R and wt Ub, and (<b>c</b>) Ub K48O and Ub K11O.</p> Full article ">Figure 9
<p>(<b>a</b>) Sequence alignment of helix <span class="html-italic">a<sub>1</sub></span>, loop L1, loop L2, and helix <span class="html-italic">a<sub>2</sub></span> of UbcH5B, UbcH7, and UbcH13 E2 enzymes, showing the amino acid conservation among the interacting with Arkadia regions. (<b>b</b>) Model of the UbcH5B–Ark RING complex (UbcH5B in light blue and Ark RING in coral). The model was prepared by superimposing Ark RING (PDBid: 2KIZ) to the X-ray structure of the UbcH5B-c-CBL complex (PDBid: 4A49) and extracting the c-CBL structure. Zn(II) ions are depicted as red spheres. (<b>c</b>) Close-up of the theoretical UbcH5B–Ark RING interface highlighting the central role of UbcH5B’s Phe62 and Ser94-Pro95-Ala96 motif. Key contact residues from Arkadia and UbcH5B are shown as coral and light blue sticks, respectively. (<b>d</b>) NMR structure of Ark RING (PDBid: 2KIZ) with Arg983 shown in orange sticks.</p> Full article ">
17 pages, 4755 KiB  
Article
2,2-Diphenethyl Isothiocyanate Enhances Topoisomerase Inhibitor-Induced Cell Death and Suppresses Multi-Drug Resistance 1 in Breast Cancer Cells
by Monika Aggarwal
Cancers 2023, 15(3), 928; https://doi.org/10.3390/cancers15030928 - 1 Feb 2023
Cited by 2 | Viewed by 2271
Abstract
We previously reported that phenethyl isothiocyanate (PEITC), a dietary-related compound, can rescue mutant p53. A structure–activity relationships study showed that the synthetic analog 2,2-diphenylethyl isothiocyanate (DPEITC) is a more potent inducer of apoptosis than natural or synthetic ITCs. Here, we showed that DPEITC [...] Read more.
We previously reported that phenethyl isothiocyanate (PEITC), a dietary-related compound, can rescue mutant p53. A structure–activity relationships study showed that the synthetic analog 2,2-diphenylethyl isothiocyanate (DPEITC) is a more potent inducer of apoptosis than natural or synthetic ITCs. Here, we showed that DPEITC inhibited the growth of triple-negative breast cancer cells (MDA-MB-231, MDA-MB-468, and Hs578T) expressing “hotspot” p53 mutants, structural (p53R280K, p53R273H) or contact (p53V157F), at IC50 values significantly lower than PEITC. DPEITC inhibited the growth of HER2+ (p53R175H SK-BR-3, p53R175H AU565) and Luminal A (p53L194F T47D) breast cancer (BC) cells harboring a p53 structural mutant. DPEITC induced apoptosis, irrespective of BC subtypes, by rescuing p53 mutants. Accordingly, the rescued p53 mutants induced apoptosis by activating canonical WT p53 targets and delaying the cell cycle. DPEITC acted synergistically with doxorubicin and camptothecin to inhibit proliferation and induce apoptosis. Under these conditions, DPEITC delayed BC cells in the G1 phase, activated p53 canonical targets, and enhanced pS1981-ATM. DPEITC reduced the expression of MDR1 and ETS1. These findings are the first report of synergism between a synthetic ITC and a chemotherapy drug via mutant p53 rescue. Furthermore, our data demonstrate that ITCs suppress the expression of cellular proteins that play a role in chemoresistance. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
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Figure 1

Figure 1
<p>DPEITC inhibits cell proliferation, induces apoptosis, and affects mutant p53 expression levels in TNBC cell lines with mutated p53. MCF7 (WT p53), MDA-MB-231 (p53<sup>R280K</sup>), MDA-MB-468 (p53<sup>R273H</sup>), and Hs578T (p53<sup>V157F</sup>) cells were treated with DMSO or DPEITC for 72 h and analyzed for percent cell proliferation (<b>A</b>) or apoptosis (<b>B</b>). (<b>C</b>) Cell lysates of TNBC cell lines with WT p53 (MCF7) or mutant p53 (MDA-MB-231, MDA-MB-468, Hs578T) treated with DMSO or the indicated concentration of DPEITC for 3 h were analyzed by Western blotting. Experiments were performed in triplicate. Error bars represent SD.</p> Full article ">Figure 2
<p>DPEITC induces a conformational change in p53 mutants, restores p53 mutant proteins’ transactivational functions, and induces G2 phase arrest in TNBC cell lines. (<b>A</b>) Immunoprecipitation to determine the effects of DPEITC on the conformation of the p53 mutant proteins, p53<sup>R280K</sup>, p53<sup>R273H</sup>, and p53<sup>V157F</sup> from MDA-MB-231, MDA-MB-468, and Hs578T cell lysates using a mutant p53 conformation-specific PAB240 antibody. The immunoprecipitated proteins were analyzed by Western blotting with p53 (FL393) antibody. The Western blot image for p53 (DO-1) serves as an input control. The blot was re-probed with an anti-GAPDH antibody. (<b>B</b>) MCF7 (WT p53), MDA-MB-468 (p53<sup>R273H</sup>), and Hs578T (p53<sup>V157F</sup>) cells were treated with DMSO or 5 μM DPEITC for 3 h and qRT-PCR of p53 canonical target genes was performed. (*** <span class="html-italic">p</span> ≤ 0.0001, ** <span class="html-italic">p</span> ≤ 0.001 and * <span class="html-italic">p</span> ≤ 0.04). (<b>C</b>) MCF7 (WT p53), MDA-MB-468 (p53<sup>R273H</sup>), and Hs578T (p53<sup>V157F</sup>) cells were treated with DMSO or 3 μM of DPEITC for 72 h and analyzed by flow cytometry. Experiments were performed in triplicate. Error bars represent SD.</p> Full article ">Figure 3
<p>Effects of DPEITC and ATZ co-treatment on cell cycle progression in TNBC cell lines. p53<sup>R280K</sup> MDA-MB-231 (<b>A</b>), p53<sup>R273H</sup> MDA-MB-468 (<b>B</b>), and WT p53 MCF7 (<b>C</b>) cells were treated with DMSO, 3 μM DPEITC, 2 mM ATZ, or 3 μM DPEITC and 2 mM ATZ for 72 h and analyzed by flow cytometry. Experiments were performed in triplicate. Error bars represent SD. Representative cell cycle images are shown.</p> Full article ">Figure 4
<p>Effects of DPEITC and ATZ co-treatment on activation of ATM in mutant p53 TNBC cells. MDA-MB-231 (p53<sup>R280K</sup>), MDA-MB-468 (p53<sup>R273H</sup>), or MCF7 (WT p53) cells were treated with DMSO, 5 μM DPEITC, 2 mM ATZ, or both for 24 h. Blots were probed using the anti-pATM S1981 antibody and re-probed with the GAPDH antibody. Experiments were performed in triplicate.</p> Full article ">Figure 5
<p>DPEITC inhibits cell proliferation, induces apoptosis, and affects p53 mutant expression levels in HER2<sub>+</sub> and Luminal A breast cancer cell lines. HER2+ SK-BR-3 (p53<sup>R175H</sup>), AU565 (p53<sup>R175H</sup>), and Luminal A T47D (p53<sup>L194F</sup>) cell lines were treated with DMSO or DPEITC for 72 h and evaluated for percent cell proliferation (<b>A</b>) or apoptosis (<b>B</b>). (<b>C</b>) Cell lysates of HER2+ and Luminal A breast cancer cell lines treated with DMSO or the indicated concentration of DPEITC for 3 h were analyzed by Western blotting. Experiments were performed in triplicate. Error bars represent SD.</p> Full article ">Figure 6
<p>DPEITC reactivates different p53 mutant proteins’ transactivational functions and induces G2 phase arrest in HER2+ and Luminal A breast cancer cell lines. (<b>A</b>) SK-BR-3 (p53<sup>R175H</sup>) and T47D (p53<sup>L194F</sup>) cells were treated with DMSO or 5 μM DPEITC for 3 h, and qRT-PCR of p53 regulated genes was performed. (*** <span class="html-italic">p</span> ≤ 0.0001 and * <span class="html-italic">p</span> ≤ 0.03). (<b>B</b>) SK-BR-3 (p53<sup>R175H</sup>) and T47D (p53<sup>L194F</sup>) cells were treated with DMSO or 5 μM of DPEITC for 72 h and analyzed by flow cytometry. Experiments were performed in triplicate. Error bars represent SD.</p> Full article ">Figure 7
<p>DPEITC enhances p53 mutant TNBC cell’s sensitivity to topoisomerase inhibitors. (<b>A</b>) MDA-MB-468 (p53<sup>R273H</sup>), MDA-MB-231 (p53<sup>R280K</sup>), or Hs578T (p53<sup>V157F</sup>) cells were treated with DPEITC, CPT, or both for 24 h. (<b>B</b>) MDA-MB-468 (p53<sup>R273H</sup>), MDA-MB-231 (p53<sup>R280K</sup>), or Hs578T (p53<sup>V157F</sup>) cells were treated with DPEITC, doxorubicin, or both for 72 h. Percent cell proliferation was determined by the WST-1 assay. Experiments were performed in triplicate. Error bars represent SD. (<b>C</b>) Effects of DPEITC on PARP1 levels. MDA-MB-468 (p53<sup>R273H</sup>) or MDA-MB-231 (p53<sup>R280K</sup>) cells were treated with DPEITC, CPT, or both for 24 h. In total, 100 μg of the cell lysate fractions were resolved by SDS-PAGE and probed with an anti-PARP antibody. Blots were stripped and re-probed with anti-GAPDH as a loading control. Experiments were performed in triplicate.</p> Full article ">Figure 8
<p>Effects of DPEITC and CPT co-treatment on cell cycle progression, activation of ATM, and canonical p53 targets. (<b>A</b>) MDA-MB-468 (p53<sup>R273H</sup>) or MDA-MB-231 (p53<sup>R280K</sup>) cells were treated with DPEITC, CPT, or both for 24 h and analyzed by flow cytometry. (<b>B</b>) MDA-MB-468 (p53<sup>R273H</sup>) or MDA-MB-231 (p53<sup>R280K</sup>) cells were treated with DPEITC, CPT, or both for 24 h. Blots were probed using the anti-pATM S1981 antibody and re-probed with the GAPDH antibody. (<b>C</b>) and (<b>D</b>) The expression of canonical p53 target genes p21 (<b>C</b>) and BAX (<b>D</b>) in MDA-MB-468 (p53<sup>R273H</sup>) or MDA-MB-231 (p53<sup>R280K</sup>) cells treated with DPEITC, CPT, or both for 24 h were analyzed by qRT-PCR. The <span class="html-italic">p</span>-values are as indicated (*** <span class="html-italic">p</span> ≤ 0.0005, ** <span class="html-italic">p</span> ≤ 0.005 and * <span class="html-italic">p</span> ≤ 0.05). Experiments were performed in triplicate. Error bars represent the SD.</p> Full article ">Figure 9
<p>DPEITC reduces the expression level of ETS1 and MDR1 in mutant p53 TNBC cell lines. MCF7 (WT p53), MDA-MB-231 (p53<sup>R280K</sup>), and MDA-MB-468 (p53<sup>R273H</sup>) were treated with 5 μM DPEITC (<b>A</b>) or 8 μM PEITC (<b>B</b>) for 24 h. Control cells were treated with DMSO. The cell lysates were resolved by SDS-PAGE, probed with anti-ETS1 and anti-MDR1 antibodies, and re-probed with anti-GAPDH antibody. Experiments were performed in triplicate.</p> Full article ">

Review

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39 pages, 10589 KiB  
Review
Human Aldehyde Dehydrogenases: A Superfamily of Similar Yet Different Proteins Highly Related to Cancer
by Vasileios Xanthis, Theodora Mantso, Anna Dimtsi, Aglaia Pappa and Vasiliki E. Fadouloglou
Cancers 2023, 15(17), 4419; https://doi.org/10.3390/cancers15174419 - 4 Sep 2023
Cited by 5 | Viewed by 3922
Abstract
The superfamily of human aldehyde dehydrogenases (hALDHs) consists of 19 isoenzymes which are critical for several physiological and biosynthetic processes and play a major role in the organism’s detoxification via the NAD(P) dependent oxidation of numerous endogenous and exogenous aldehyde substrates to their [...] Read more.
The superfamily of human aldehyde dehydrogenases (hALDHs) consists of 19 isoenzymes which are critical for several physiological and biosynthetic processes and play a major role in the organism’s detoxification via the NAD(P) dependent oxidation of numerous endogenous and exogenous aldehyde substrates to their corresponding carboxylic acids. Over the last decades, ALDHs have been the subject of several studies as it was revealed that their differential expression patterns in various cancer types are associated either with carcinogenesis or promotion of cell survival. Here, we attempt to provide a thorough review of hALDHs’ diverse functions and 3D structures with particular emphasis on their role in cancer pathology and resistance to chemotherapy. We are especially interested in findings regarding the association of structural features and their changes with effects on enzymes’ functionalities. Moreover, we provide an updated outline of the hALDHs inhibitors utilized in experimental or clinical settings for cancer therapy. Overall, this review aims to provide a better understanding of the impact of ALDHs in cancer pathology and therapy from a structural perspective. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
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Figure 1

Figure 1
<p>The structure of mitochondrial hALDH1B1 monomer in complex with the NAD (Nicotinamide Adenine Dinucleotide) cofactor and the ZGG (8-(2-methoxyphenyl)-10-(4-phenylphenyl)-1,8-diazabicyclo[5.3.0]deca-1(7),9-diene) inhibitor (PDBid:7MJD). The protein is shown in cartoon representation. Cofactor and inhibitor are shown as space-filling models and indicate the substrate and cofactor binding sites on the protein, respectively. The three catalytic residues, i.e., Cys 302, Glu 268, and Asn 169, are shown with spheres and indicate the protein’s active site. The monomer’s structure consists of a catalytic domain (pink), a cofactor binding domain (blue), and an oligomerization domain (green). (<b>A</b>–<b>C</b>) Three views of the monomer related with 90-degree rotations around the indicated axis. The zoom-in view of (<b>B</b>) focuses on the NAD and ZGG binding sites and thus highlights the spatial proximity of cofactor and substrate binding sites on the protein’s structure. The catalytic (<b>D</b>) and cofactor (<b>E</b>) binding domains fold as Rossmann motifs. In the interface of catalytic and cofactor binding domains, the active site of the enzyme is located.</p> Full article ">Figure 2
<p>Multiple sequence alignment of the 19 hALDHs. The part of the alignment which includes the catalytic triad (highlighted in blue) is shown. The alignment shows that all hALDHs but ALDH16A1, ALDH6A1, and ALDH18A1 have in common a catalytic triad consisting of a cysteine (catalytic thiol), a glutamic acid (general base), and an asparagine (residue important for stabilizing the reaction’s intermediate). ALDH6A1 follows a slightly different mechanism compared with the other members of the superfamily (see text) and possesses a slightly modified catalytic triad where glutamic acid has been substituted by an asparagine, while the other two catalytic residues (cysteine and asparagine) are conserved. ALDH18A1 is the most distant member of the superfamily (see text and <a href="#cancers-15-04419-f003" class="html-fig">Figure 3</a>) which is also evident from the fact that its active site incorporates only the catalytic cysteine. Last, ALDH16A1 is a pseudoenzyme without enzymatic activity and includes none of the catalytic residues.</p> Full article ">Figure 3
<p>All against all structure analysis of hALDHs with determined 3D structures through DALI server [<a href="#B30-cancers-15-04419" class="html-bibr">30</a>]. Protein names and the PDB codes of the structures used as representatives of each protein for the analysis are shown together. (<b>A</b>) Heatmap of structural similarity matrix based on Dali Z-scores. (<b>B</b>) Structural similarity dendrogram. The dendrogram is derived by average linkage clustering of the structural similarity matrix (Dali Z-scores).The dendrogram figure was prepared with the iTOL tool [<a href="#B31-cancers-15-04419" class="html-bibr">31</a>].</p> Full article ">Figure 4
<p>Topology diagrams of the three domains for each of the 12 hALDHs of known structure. All three domains of hALDH18A1 are distinct, while all the other members have in common at least the topology of catalytic domain. hALDH3A1 and 3A2 have in addition a common topology of their cofactor binding domains. ALDH4A1 shares with the hALDH3Ai pair a quite common oligomerization domain. hALDH1/2, 5A1, and 9A1 have common topologies in all three domains, while hALDH4A1 and 7A1 might be the bridge between the 3Ai subgroup and the 1/2, 5A1, 9A1 subgroup.</p> Full article ">Figure 5
<p>Dimer organization—from three different views, 90 degrees apart—for three representative structures of hALDH superfamily. The structures are shown with surface representation and the colours indicate the catalytic (pink), cofactor binding (blue), and oligomerization (green) domains. (<b>A</b>) hALDH3A1 (PDBid: 3SZB). (<b>B</b>) hALDH1A1 (PDBid: 4WB9). (<b>C</b>) hALDH18A1 (PDBid: 2H5G).</p> Full article ">Figure 6
<p>Representative examples of hALDH quaternary assemblies. Each structure is shown from three different views, as indicated. Space-filling models are used, and the different colours represent different monomers. (<b>A</b>) hALDH3A1 dimer (PDBid: 3SZB). (<b>B</b>) hALDH1A1 tetramer (PDBid: 4WB9). (<b>C</b>) hALDH5A1 dodecamer of reduced protein (PDBid: 2W8O).</p> Full article ">Figure 7
<p>Diagram presenting the multiple roles of ALDHs.</p> Full article ">Figure 8
<p>Location of Lysine 353 on the surface of ALDH1A1 tetramer and how its acetylation imposes steric hindrance and affects its catalytic ability. (<b>A</b>,<b>B</b>) Cartoon representation of hALDH1A1. Each monomer is shown with a different colour. Lysine 353 (gray space-filling model) is found on the rim of NAD(H) (yellow space-filling model) binding pocket. (<b>C</b>,<b>D</b>) Zoom in on the Lysine 353/NADH binding area. Lysine is shown in a gray sticks model superimposed with van der Waals dots of non-hydrogen atoms. In (<b>C</b>), the unmodified Lys makes optimum interactions with the bound NADH. (<b>D</b>) A model of acetylated Lys353 shows how the extra acetyl-group restricts the available space and makes unfavourable the NADH binding.</p> Full article ">Figure 9
<p>Non-specific inhibitors in complex with hALDHs. (<b>A</b>) hALDH2/Aldi-3 complex (PDBid: 3SZ9). The inhibitor is inside the active site and forms a covalent bond with the catalytic cysteine. (<b>B</b>) hALDH3A1/Aldi-1 complex (PDBid: 3SZB). (<b>C</b>) hALDH7A1/DEAB complex (PDBid: 4X0T).</p> Full article ">Figure 10
<p>Specific inhibitors in complex with hALDHs. Proteins are shown in cartoon representation and residues significant for the inhibitor-protein interactions are shown with sticks. Especially, non-conserved driving-specificity residues are shown in spacefill. Protein elements are all in green. Inhibitors are represented with gray-carbon stick models. (<b>A</b>) hALDH1A1/CM026 complex (PDBid:4WP7). (<b>B</b>) hALDH1A1/CM037 complex (PDBid:4X4L). (<b>C</b>) hALDH1A3/NR6 complex (PDBid:7A6Q). (<b>D</b>) hALDH1A3/MCI-INI-3 complex (PDBid:6TGW). (<b>E</b>) hALDH3A1/CB29 complex (PDBid:4H80). (<b>F</b>) hALDH3A1/CB7 complex (PDBid:4L2O).</p> Full article ">Figure 11
<p>A small window from the sequence alignment of hALDHs, which highlights two residues experimentally characterized as driving-specificity residues for designing inhibitors. Gly458 for ALDH1A1 (last residue of the horizontal yellow highlight) and Tyr472 for ALDH1A3 (last residue of the horizontal pink highlight). For more details, see text and <a href="#cancers-15-04419-f010" class="html-fig">Figure 10</a>.</p> Full article ">
20 pages, 2193 KiB  
Review
The Structural Dynamics, Complexity of Interactions, and Functions in Cancer of Multi-SAM Containing Proteins
by Christopher M. Clements, Morkos A. Henen, Beat Vögeli and Yiqun G. Shellman
Cancers 2023, 15(11), 3019; https://doi.org/10.3390/cancers15113019 - 1 Jun 2023
Cited by 2 | Viewed by 1871
Abstract
SAM domains are crucial mediators of diverse interactions, including those important for tumorigenesis or metastasis of cancers, and thus SAM domains can be attractive targets for developing cancer therapies. This review aims to explore the literature, especially on the recent findings of the [...] Read more.
SAM domains are crucial mediators of diverse interactions, including those important for tumorigenesis or metastasis of cancers, and thus SAM domains can be attractive targets for developing cancer therapies. This review aims to explore the literature, especially on the recent findings of the structural dynamics, regulation, and functions of SAM domains in proteins containing more than one SAM (multi-SAM containing proteins, MSCPs). The topics here include how intrinsic disorder of some SAMs and an additional SAM domain in MSCPs increase the complexity of their interactions and oligomerization arrangements. Many similarities exist among these MSCPs, including their effects on cancer cell adhesion, migration, and metastasis. In addition, they are all involved in some types of receptor-mediated signaling and neurology-related functions or diseases, although the specific receptors and functions vary. This review also provides a simple outline of methods for studying protein domains, which may help non-structural biologists to reach out and build new collaborations to study their favorite protein domains/regions. Overall, this review aims to provide representative examples of various scenarios that may provide clues to better understand the roles of SAM domains and MSCPs in cancer in general. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
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Figure 1
<p>Domain architecture of MSCP proteins and families. Proteins are aligned by final SAM domain. Sizes are relative and not to scale. Representative protein members are shown here for each family. Stars indicate SAM domains with experimentally shown disordered states.</p> Full article ">Figure 2
<p>Binding of the disordered SAM domain in tandem with a well-folded SAM domain leads to a heightened level of complexity in the system. Schematic representation illustrating the potential impact of having two SAM domains in tandem. In isolation, the disordered SAM domain (domain A: depicted in red) exhibits higher conformational flexibility. However, upon interacting with a folded SAM domain (domain B: illustrated in blue), a dimer is formed, accompanied by folding of the disordered domain. This event may result in the formation of higher-order polymeric structures crucial for the functioning of the system.</p> Full article ">Figure 3
<p>Diversity in multimerizations of the MSCPs’ SAMs. (<b>A</b>) The ANKS1B tandem SAM1/2 domain (PDB: 2KIV) forms an intramolecular head-to-tail monomer. SAM2 is disordered when not bound to SAM1, which may serve to regulate the nuclear localization sequence (yellow) in helix 5 of SAM2. (<b>B</b>) Liprin-α and Liprin-β each has three tandem SAM domains that intramolecularly trimerize (PDB: 3TAD). SAM3 of Liprin-β can dimerize with SAM1 of Liprin-α, creating a Liprin heterodimer and a linear SAM hexamer in a head-to-tail fashion. (<b>C</b>) CASKIN1 tandem SAM1/2 (PDB: 3SEN) forms intramolecular head-to-tail interactions that then extend to other CASKIN2 tandem SAM1/2 domains to form a helical oligomer of monomers. (<b>D</b>) The tandem SAM1/2 domain of CASKIN2 (PDB: 5L1M) forms intermolecular interactions to form a dimer that may expand to an oligomer of dimers. This “domain-swapped dimer” can lead to a branched oligomer in contrast to CASKIN1′s spiral oligomer. (<b>E</b>) The SARM1 tandem SAM1/2 (PDB: 6QWV) form intramolecular head-to-tail interactions that then utilize lateral interactions to form a stacked closed octameric ring.</p> Full article ">Figure 4
<p>Schematic for approaching the study of MSCP SAM domains. The numbers at the end indicate the subsections.</p> Full article ">Figure 5
<p>The Alpha-Fold predicted structure of SASH1′s <span class="html-italic">C</span>-terminal (1082–1247). This includes the SAM2 domain (1177–1241; red) and a novel SAM-like helical bundle (1092–1170; cyan).</p> Full article ">
12 pages, 678 KiB  
Review
Mining the Immunopeptidome for Antigenic Peptides in Cancer
by Ricardo A. León-Letelier, Hiroyuki Katayama and Sam Hanash
Cancers 2022, 14(20), 4968; https://doi.org/10.3390/cancers14204968 - 11 Oct 2022
Cited by 11 | Viewed by 5061
Abstract
Although harnessing the immune system for cancer therapy has shown success, response to immunotherapy has been limited. The immunopeptidome of cancer cells presents an opportunity to discover novel antigens for immunotherapy applications. These neoantigens bind to MHC class I and class II molecules. [...] Read more.
Although harnessing the immune system for cancer therapy has shown success, response to immunotherapy has been limited. The immunopeptidome of cancer cells presents an opportunity to discover novel antigens for immunotherapy applications. These neoantigens bind to MHC class I and class II molecules. Remarkably, the immunopeptidome encompasses protein post-translation modifications (PTMs) that may not be evident from genome or transcriptome profiling. A case in point is citrullination, which has been demonstrated to induce a strong immune response. In this review, we cover how the immunopeptidome, with a special focus on PTMs, can be utilized to identify cancer-specific antigens for immunotherapeutic applications. Full article
(This article belongs to the Special Issue Protein Structure and Cancer)
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<p>Post-translationally modified peptide-based cancer vaccine workflow. The figure depicts cancer cell antigen processing of intracellular and extracellular proteins, subsequently as peptides bound to MHC-I or MHC-II. Some of the proteins have PTMs in their structure, which are sketched in colors (citrullination: red; phosphorylation: blue; glycosylation: green) as well as in MHC-bound peptides. The MHC-bound peptides are identified by means of liquid chromatography-mass spectrometry (LC/MS), to derive the cancer cell immunopeptidome. From the immunopeptidome data, peptides with PTMs can be selected as antigens for cancer vaccines.</p> Full article ">
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