Unveiling Protein Functions, Dynamics and Interactions in Health and Disease Using Experimental and Theoretical Approaches

A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Bioinformatics and Systems Biology".

Deadline for manuscript submissions: closed (30 June 2024) | Viewed by 30685

Special Issue Editors


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Guest Editor
1. Department of Life and Environmental Sciences, New York-Marche Structural Biology Centre (NY-MaSBiC), Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy
2. Neuronal Death and Neuroprotection Unit, Department of Neuroscience, Mario Negri Institute for Pharmacological Research-IRCCS, Via Mario Negri 2, 20156 Milan, Italy
3. National Biodiversity Future Center (NBFC), Palermo, Italy
Interests: protein-protein interactions; molecular biology; structural bioinformatics; computational chemistry; bioinformatics; neuroscience; biosensors development

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Co-Guest Editor
Dipartimento di Scienze della Vita e dell’Ambiente (DiSVA), Università Politecnica delle Marche, 60131 Ancona, Italy
Interests: specialized in enzymology and biochemistry of protein synthesis; assay development (in vivo expression, enzymatic activity, UV-vis fluorescence spectroscopy); biochemistry of bacterial translation factors; expression analysis of human eIF5A-2 isoform

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Co-Guest Editor
1. Department of Life and Environmental Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy
2. New York-Marche Structural Biology Center (NY-MaSBiC), Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancona, Italy
Interests: digestion and cloning of plasmids; bacterial transformation, recombinant protein expression by bacterial; archaeal and mammalian culture; electrophoretic separation on polyacrylamide gels; western blot, dot-blot; protein purification with chromatographic separation (AKTA, HPLC, FPLC) based on affinity; ion-exchange and size exclusion; high throughput screening; genomic DNA isolation; total RNA isolation; in vitro transcription; sucrose and glycerol gradients preparation; ribosome profiling, protein and RNA immunoprecipitation; PCR and reverse-transcriptase-PCR (RT-PCR); zymogram assay; RNA cleavage assay, radiolabeled of RNA; electrophoresis mobility shift assay (EMSA); pull-down assay

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Co-Guest Editor
Department of Earth and Environmental Sciences, University of Milano-Bicocca, 20126 Milan, Italy
Interests: molecular dynamics; molecular dynamics simulations; metadynamics; ligand binding

Special Issue Information

Dear Colleagues,

In the last years, important scientific results have been obtained thanks to the application of approaches and techniques coming from the most diverse branches of science, sometimes very far from each other. This is particularly true for the biological sciences, in which, for example, the use of computational techniques to support experimental data, and vice versa, has been a fruitful strategy in a large number of studies. In this scenario, the study of proteins features such as the interplay between their structure/dynamics and function, and their intermolecular interactions, both in health and diseases, acquires a key role.

Frequently, the sole experimental results, even if combining different disciplines, do not provide a clear understanding of a complex biological problem. In particular, in the study of proteins function, some key questions can arise, for instance, concerning the role of mutations or of conformational changes of proteins structure associated with a specific cellular function. The use of standard or advanced theoretical approaches (i.e., classical molecular dynamics, molecular docking or enhanced sampling approaches) can help both the comprehension of experimental data or, in turn, can better guide the design of future experiments.

The aim of this Special Issue is to bridge the gap sometimes existing among different approaches that are exploited for a common purpose, i.e., understanding and describing different functional-dynamical features of proteins in specific physiological and pathological conditions. A multi-disciplinary collection including molecular biology, structural biology, biophysics, biochemistry, bioinformatics and computational chemistry is the scope of this issue.

In this context, this collection aims at reporting current experimental and computational trends in the field of proteins functions, interactions and dynamics. In this Special Issue, original research articles, Reviews, and Perspectives are welcome. Research areas may include (but not limited to) the following:

(i) Understanding the structural, dynamical and functional role of point mutations

(ii) Conformational changes of proteins linked to their function

(iii) Protein-proteins, protein-nucleic acids and protein-ligand interactions in health and diseases

(iv) Protein design and engineering for biomedical applications

I look forward to receiving your contributions.

Dr. Daniele Di Marino
Dr. Cristina Maracci
Dr. Alice Romagnoli
Dr. Stefano Motta
Guest Editors

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Keywords

  • Protein’s structure and dynamics
  • Protein-protein interactions
  • Point mutations
  • Molecular biology
  • Molecular modelling
  • Molecular dynamics
  • Biochemistry
  • Bioinformatics
  • Protein-ligand interaction
  • Protein-nucleic acids interaction
  • Protein’s conformational changes

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Published Papers (9 papers)

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Research

17 pages, 28499 KiB  
Article
An Overview of Two Old Friends Associated with Platelet Redox Signaling, the Protein Disulfide Isomerase and NADPH Oxidase
by Andrés Trostchansky and Marcelo Alarcon
Biomolecules 2023, 13(5), 848; https://doi.org/10.3390/biom13050848 - 17 May 2023
Cited by 1 | Viewed by 1763
Abstract
Oxidative stress participates at the baseline of different non-communicable pathologies such as cardiovascular diseases. Excessive formation of reactive oxygen species (ROS), above the signaling levels necessary for the correct function of organelles and cells, may contribute to the non-desired effects of oxidative stress. [...] Read more.
Oxidative stress participates at the baseline of different non-communicable pathologies such as cardiovascular diseases. Excessive formation of reactive oxygen species (ROS), above the signaling levels necessary for the correct function of organelles and cells, may contribute to the non-desired effects of oxidative stress. Platelets play a relevant role in arterial thrombosis, by aggregation triggered by different agonists, where excessive ROS formation induces mitochondrial dysfunction and stimulate platelet activation and aggregation. Platelet is both a source and a target of ROS, thus we aim to analyze both the platelet enzymes responsible for ROS generation and their involvement in intracellular signal transduction pathways. Among the proteins involved in these processes are Protein Disulphide Isomerase (PDI) and NADPH oxidase (NOX) isoforms. By using bioinformatic tools and information from available databases, a complete bioinformatic analysis of the role and interactions of PDI and NOX in platelets, as well as the signal transduction pathways involved in their effects was performed. We focused the study on analyzing whether these proteins collaborate to control platelet function. The data presented in the current manuscript support the role that PDI and NOX play on activation pathways necessary for platelet activation and aggregation, as well as on the platelet signaling imbalance produced by ROS production. Our data could be used to design specific enzyme inhibitors or a dual inhibition for these enzymes with an antiplatelet effect to design promising treatments for diseases involving platelet dysfunction. Full article
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22 pages, 3930 KiB  
Article
Structural–Functional Relationship of the Ribonucleolytic Activity of aIF5A from Sulfolobus solfataricus
by Alice Romagnoli, Paolo Moretti, Mattia D’Agostino, Jesmina Rexha, Nunzio Perta, Astra Piccinini, Daniele Di Marino, Francesco Spinozzi and Anna La Teana
Biomolecules 2022, 12(10), 1432; https://doi.org/10.3390/biom12101432 - 6 Oct 2022
Cited by 2 | Viewed by 1937
Abstract
The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, [...] Read more.
The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, we have undertaken a structural and functional characterization of aIF5A from the crenarchaeal Sulfolobus solfataricus model organism. We confirm the association of aIF5A with several RNA molecules in vivo and demonstrate that the protein is endowed with a ribonuclease activity which is specific for long and structured RNA. By means of biochemical and structural approaches we show that aIF5A can exist in both monomeric and dimeric conformations and the monomer formation is favored by the association with RNA. Finally, modelling of the three-dimensional structure of S. solfataricus aIF5A shows an extended positively charged surface which may explain its strong tendency to associate to RNA in vivo. Full article
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11 pages, 4149 KiB  
Article
Benchmarking the Accuracy of AlphaFold 2 in Loop Structure Prediction
by Amy O. Stevens and Yi He
Biomolecules 2022, 12(7), 985; https://doi.org/10.3390/biom12070985 - 14 Jul 2022
Cited by 36 | Viewed by 5310
Abstract
The inhibition of protein–protein interactions is a growing strategy in drug development. In addition to structured regions, many protein loop regions are involved in protein–protein interactions and thus have been identified as potential drug targets. To effectively target such regions, protein structure is [...] Read more.
The inhibition of protein–protein interactions is a growing strategy in drug development. In addition to structured regions, many protein loop regions are involved in protein–protein interactions and thus have been identified as potential drug targets. To effectively target such regions, protein structure is critical. Loop structure prediction is a challenging subgroup in the field of protein structure prediction because of the reduced level of conservation in protein sequences compared to the secondary structure elements. AlphaFold 2 has been suggested to be one of the greatest achievements in the field of protein structure prediction. The AlphaFold 2 predicted protein structures near the X-ray resolution in the Critical Assessment of protein Structure Prediction (CASP 14) competition in 2020. The purpose of this work is to survey the performance of AlphaFold 2 in specifically predicting protein loop regions. We have constructed an independent dataset of 31,650 loop regions from 2613 proteins (deposited after the AlphaFold 2 was trained) with both experimentally determined structures and AlphaFold 2 predicted structures. With extensive evaluation using our dataset, the results indicate that AlphaFold 2 is a good predictor of the structure of loop regions, especially for short loop regions. Loops less than 10 residues in length have an average Root Mean Square Deviation (RMSD) of 0.33 Å and an average the Template Modeling score (TM-score) of 0.82. However, we see that as the number of residues in a given loop increases, the accuracy of AlphaFold 2’s prediction decreases. Loops more than 20 residues in length have an average RMSD of 2.04 Å and an average TM-score of 0.55. Such a correlation between accuracy and length of the loop is directly linked to the increase in flexibility. Moreover, AlphaFold 2 does slightly over-predict α-helices and β-strands in proteins. Full article
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11 pages, 3573 KiB  
Article
NAMPT Inhibitor and P73 Activator Represses P53 R175H Mutated HNSCC Cell Proliferation in a Synergistic Manner
by Bi-He Cai, Zhi-Yu Bai, Ching-Feng Lien, Si-Jie Yu, Rui-Yu Lu, Ming-Han Wu, Wei-Chen Wu, Chia-Chi Chen and Yi-Chiang Hsu
Biomolecules 2022, 12(3), 438; https://doi.org/10.3390/biom12030438 - 12 Mar 2022
Cited by 9 | Viewed by 3202
Abstract
The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain [...] Read more.
The p53 family has the following three members: p53, p63 and p73. p53 is a tumor suppressor gene that frequently exhibits mutation in head and neck cancer. Most p53 mutants are loss-of-function (LoF) mutants, but some acquire some oncogenic function, such as gain of function (GoF). It is known that the aggregation of mutant p53 can induce p53 GoF. The p73 activators RETRA and NSC59984 have an anti-cancer effect in p53 mutation cells, but we found that p73 activators were not effective in all head and neck squamous cell carcinoma (HNSCC) cell lines, with different p53 mutants. A comparison of the gene expression profiles of several regulator(s) in mutant HNSCC cells with or without aggregation of p53 revealed that nicotinamide phosphoribosyltransferase (NAMPT) is a key regulator of mutant p53 aggregation. An NAMPT inhibitor, to reduce abnormal aggregation of mutant p53, used in combination with a p73 activator, was able to effectively repress growth in HNSCC cells with p53 GoF mutants. This study, therefore, suggests a potential combination therapy approach for HNSCC with a p53 GoF mutation. Full article
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16 pages, 5840 KiB  
Article
A Computational Approach to Investigate TDP-43 RNA-Recognition Motif 2 C-Terminal Fragments Aggregation in Amyotrophic Lateral Sclerosis
by Greta Grassmann, Mattia Miotto, Lorenzo Di Rienzo, Federico Salaris, Beatrice Silvestri, Elsa Zacco, Alessandro Rosa, Gian Gaetano Tartaglia, Giancarlo Ruocco and Edoardo Milanetti
Biomolecules 2021, 11(12), 1905; https://doi.org/10.3390/biom11121905 - 19 Dec 2021
Cited by 6 | Viewed by 3397
Abstract
Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the aggregate-associated diseases, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. In fact, although understanding the processes that cause the disease is [...] Read more.
Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the aggregate-associated diseases, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. In fact, although understanding the processes that cause the disease is still an open challenge, its relationship with protein aggregation is widely known. In particular, human TDP-43, an RNA/DNA binding protein, is a major component of the pathological cytoplasmic inclusions observed in ALS patients. Indeed, the deposition of the phosphorylated full-length TDP-43 in spinal cord cells has been widely studied. Moreover, it has also been shown that the brain cortex presents an accumulation of phosphorylated C-terminal fragments (CTFs). Even if it is debated whether the aggregation of CTFs represents a primary cause of ALS, it is a hallmark of TDP-43 related neurodegeneration in the brain. Here, we investigate the CTFs aggregation process, providing a computational model of interaction based on the evaluation of shape complementarity at the molecular interfaces. To this end, extensive Molecular Dynamics (MD) simulations were conducted for different types of protein fragments, with the aim of exploring the equilibrium conformations. Adopting a newly developed approach based on Zernike polynomials, able to find complementary regions in the molecular surface, we sampled a large set of solvent-exposed portions of CTFs structures as obtained from MD simulations. Our analysis proposes and assesses a set of possible association mechanisms between the CTFs, which could drive the aggregation process of the CTFs. To further evaluate the structural details of such associations, we perform molecular docking and additional MD simulations to propose possible complexes and assess their stability, focusing on complexes whose interacting regions are both characterized by a high shape complementarity and involve β3 and β5 strands at their interfaces. Full article
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29 pages, 5664 KiB  
Article
Comprehensive Characterization of the Coding and Non-Coding Single Nucleotide Polymorphisms in the Tumor Protein p63 (TP63) Gene Using In Silico Tools
by Shamima Akter, Shafaat Hossain, Md. Ackas Ali, Md. Ismail Hosen and Hossain Uddin Shekhar
Biomolecules 2021, 11(11), 1733; https://doi.org/10.3390/biom11111733 - 20 Nov 2021
Cited by 2 | Viewed by 3982
Abstract
Single nucleotide polymorphisms (SNPs) help to understand the phenotypic variations in humans. Genome-wide association studies (GWAS) have identified SNPs located in the tumor protein 63 (TP63) locus to be associated with the genetic susceptibility of cancers. However, there is a lack of in-depth [...] Read more.
Single nucleotide polymorphisms (SNPs) help to understand the phenotypic variations in humans. Genome-wide association studies (GWAS) have identified SNPs located in the tumor protein 63 (TP63) locus to be associated with the genetic susceptibility of cancers. However, there is a lack of in-depth characterization of the structural and functional impacts of the SNPs located at the TP63 gene. The current study was designed for the comprehensive characterization of the coding and non-coding SNPs in the human TP63 gene for their functional and structural significance. The functional and structural effects of the SNPs were investigated using a wide variety of computational tools and approaches, including molecular dynamics (MD) simulation. The deleterious impact of eight nonsynonymous SNPs (nsSNPs) affecting protein stability, structure, and functions was measured by using 13 bioinformatics tools. These eight nsSNPs are in highly conserved positions in protein and were predicted to decrease protein stability and have a deleterious impact on the TP63 protein function. Molecular docking analysis showed five nsSNPs to reduce the binding affinity of TP63 protein to DNA with significant results for three SNPs (R319H, G349E, and C347F). Further, MD simulations revealed the possible disruption of TP63 and DNA binding, hampering the essential protein function. PolymiRTS study found five non-coding SNPs in miRNA binding sites, and the GTEx portal recognized five eQTLs SNPs in single tissue of the lung, heart (LV), and cerebral hemisphere (brain). Characterized nsSNPs and non-coding SNPs will help researchers to focus on TP63 gene loci and ascertain their association with certain diseases. Full article
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15 pages, 1950 KiB  
Article
DIPEND: An Open-Source Pipeline to Generate Ensembles of Disordered Segments Using Neighbor-Dependent Backbone Preferences
by Zita Harmat, Dániel Dudola and Zoltán Gáspári
Biomolecules 2021, 11(10), 1505; https://doi.org/10.3390/biom11101505 - 12 Oct 2021
Cited by 3 | Viewed by 2240
Abstract
Ensemble-based structural modeling of flexible protein segments such as intrinsically disordered regions is a complex task often solved by selection of conformers from an initial pool based on their conformity to experimental data. However, the properties of the conformational pool are crucial, as [...] Read more.
Ensemble-based structural modeling of flexible protein segments such as intrinsically disordered regions is a complex task often solved by selection of conformers from an initial pool based on their conformity to experimental data. However, the properties of the conformational pool are crucial, as the sampling of the conformational space should be sufficient and, in the optimal case, relatively uniform. In other words, the ideal sampling is both efficient and exhaustive. To achieve this, specialized tools are usually necessary, which might not be maintained in the long term, available on all platforms or flexible enough to be tweaked to individual needs. Here, we present an open-source and extendable pipeline to generate initial protein structure pools for use with selection-based tools to obtain ensemble models of flexible protein segments. Our method is implemented in Python and uses ChimeraX, Scwrl4, Gromacs and neighbor-dependent backbone distributions compiled and published previously by the Dunbrack lab. All these tools and data are publicly available and maintained. Our basic premise is that by using residue-specific, neighbor-dependent Ramachandran distributions, we can enhance the efficient exploration of the relevant region of the conformational space. We have also provided a straightforward way to bias the sampling towards specific conformations for selected residues by combining different conformational distributions. This allows the consideration of a priori known conformational preferences such as in the case of preformed structural elements. The open-source and modular nature of the pipeline allows easy adaptation for specific problems. We tested the pipeline on an intrinsically disordered segment of the protein Cd3ϵ and also a single-alpha helical (SAH) region by generating conformational pools and selecting ensembles matching experimental data using the CoNSEnsX+ server. Full article
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10 pages, 2461 KiB  
Article
Molecular Dynamics Simulation Study of the Interaction between Human Angiotensin Converting Enzyme 2 and Spike Protein Receptor Binding Domain of the SARS-CoV-2 B.1.617 Variant
by Priya Antony and Ranjit Vijayan
Biomolecules 2021, 11(8), 1244; https://doi.org/10.3390/biom11081244 - 20 Aug 2021
Cited by 19 | Viewed by 3727
Abstract
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had a significant impact on people’s daily lives. The rapidly spreading B.1.617 lineage harbors two key mutations—L452R and E484Q—in the receptor binding domain (RBD) of its spike (S) protein. To understand the impact and [...] Read more.
The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had a significant impact on people’s daily lives. The rapidly spreading B.1.617 lineage harbors two key mutations—L452R and E484Q—in the receptor binding domain (RBD) of its spike (S) protein. To understand the impact and structural dynamics of the variations in the interface of S protein and its host factor, the human angiotensin-converting enzyme 2 (hACE2), triplicate 500 ns molecular dynamics simulations were performed using single (E484Q or L452R) and double (E484Q + L452R) mutant structures and compared to wild type simulations. Our results indicate that the E484Q mutation disrupts the conserved salt bridge formed between Lys31 of hACE2 and Glu484 of S protein. Additionally, E484Q, which could favor the up conformation of the RBD, may help in enhanced hACE2 binding and immune escape. L452R introduces a charged patch near the binding surface that permits increased electrostatic attraction between the proteins. An improved network of intramolecular interactions observed is likely to increase the stability of the S protein and conformational changes may prevent the binding of neutralizing antibodies. The results obtained from the molecular dynamics simulations suggest that structural and dynamic changes introduced by these variations enhance the affinity of the viral S protein to hACE2 and could form the basis for further studies. Full article
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20 pages, 2926 KiB  
Article
Pharmacokinetics and Molecular Modeling Indicate nAChRα4-Derived Peptide HAEE Goes through the Blood–Brain Barrier
by Yurii A. Zolotarev, Vladimir A. Mitkevich, Stanislav I. Shram, Alexei A. Adzhubei, Anna P. Tolstova, Oleg B. Talibov, Alexander K. Dadayan, Nikolai F. Myasoyedov, Alexander A. Makarov and Sergey A. Kozin
Biomolecules 2021, 11(6), 909; https://doi.org/10.3390/biom11060909 - 18 Jun 2021
Cited by 5 | Viewed by 2967
Abstract
One of the treatment strategies for Alzheimer’s disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aβ) and blocking its aggregation in the brain. Previously, we found that intravenous administration of the synthetic tetrapeptide Acetyl-His-Ala-Glu-Glu-Amide (HAEE), which [...] Read more.
One of the treatment strategies for Alzheimer’s disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aβ) and blocking its aggregation in the brain. Previously, we found that intravenous administration of the synthetic tetrapeptide Acetyl-His-Ala-Glu-Glu-Amide (HAEE), which is an analogue of the 35–38 region of the α4 subunit of α4β2 nicotinic acetylcholine receptor and specifically binds to the 11–14 site of Aβ, reduced the development of cerebral amyloidogenesis in a mouse model of AD. In the current study on three types of laboratory animals, we determined the biodistribution and tissue localization patterns of HAEE peptide after single intravenous bolus administration. The pharmacokinetic parameters of HAEE were established using uniformly tritium-labeled HAEE. Pharmacokinetic data provided evidence that HAEE goes through the blood–brain barrier. Based on molecular modeling, a role of LRP1 in receptor-mediated transcytosis of HAEE was proposed. Altogether, the results obtained indicate that the anti-amyloid effect of HAEE, previously found in a mouse model of AD, most likely occurs due to its interaction with Aβ species directly in the brain. Full article
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