The complex impact of cancer-related missense mutations on the stability and on the biophysical and biochemical properties of MAPK1 and MAPK3 somatic variants.

Mitogen-activated protein kinases 1 and 3 (MAPK1 and MAPK3), also called extracellular regulated kinases (ERK2 and ERK1), are serine/threonine kinase activated downstream by the Ras/Raf/MEK/ERK signal transduction cascade that regulates a variety of cellular processes. A dysregulation of MAPK cascade is frequently associated to missense mutations on its protein components and may be related to many pathologies, including cancer. In this study we selected from COSMIC database a set of MAPK1 and MAPK3 somatic variants found in cancer tissues carrying missense mutations distributed all over the MAPK1 and MAPK3 sequences. The proteins were expressed as pure recombinant proteins, and their biochemical and biophysical properties have been studied in comparison with the wild type. The missense mutations lead to changes in the tertiary arrangements of all the variants. The thermodynamic stability of the wild type and variants has been investigated in the non-phosphorylated and in the phosphorylated form. Significant differences in the thermal stabilities of most of the variants have been observed, as well as changes in the catalytic efficiencies. The energetics of the catalytic reaction is affected for all the variants for both the MAPK proteins. The stability changes and the variation in the enzyme catalysis observed for most of MAPK1/3 variants suggest that a local change in a residue, distant from the catalytic site, may have long-distance effects that reflect globally on enzyme stability and functions.

Analysis and Interpretation of the Impact of Missense Variants in Cancer.

Large scale genome sequencing allowed the identification of a massive number of genetic variations, whose impact on human health is still unknown. In this review we analyze, by an in silico-based strategy, the impact of missense variants on cancer-related genes, whose effect on protein stability and function was experimentally determined. We collected a set of 164 variants from 11 proteins to analyze the impact of missense mutations at structural and functional levels, and to assess the performance of state-of-the-art methods (FoldX and Meta-SNP) for predicting protein stability change and pathogenicity. The result of our analysis shows that a combination of experimental data on protein stability and in silico pathogenicity predictions allowed the identification of a subset of variants with a high probability of having a deleterious phenotypic effect, as confirmed by the significant enrichment of the subset in variants annotated in the COSMIC database as putative cancer-driving variants. Our analysis suggests that the integration of experimental and computational approaches may contribute to evaluate the risk for complex disorders and develop more effective treatment strategies.

A Glimpse into the Structural Properties of the Intermediate and Transition State in the Folding of Bromodomain 2 Domain 2 by Φ Value Analysis.

Bromodomains (BRDs) are small protein interaction modules of about 110 amino acids that selectively recognize acetylated lysine in histones and other proteins. These domains have been identified in a variety of multi-domain proteins involved in transcriptional regulation or chromatin remodeling in eukaryotic cells. BRD inhibition is considered an attractive therapeutic approach in epigenetic disorders, particularly in oncology. Here, we present a Φ value analysis to investigate the folding pathway of the second domain of BRD2 (BRD2(2)). Using an extensive mutational analysis based on 25 site-directed mutants, we provide structural information on both the intermediate and late transition state of BRD2(2). The data reveal that the C-terminal region represents part of the initial folding nucleus, while the N-terminal region of the domain consolidates its structure only later in the folding process. Furthermore, only a small number of native-like interactions have been identified, suggesting the presence of a non-compact, partially folded state with scarce native-like characteristics. Taken together, these results indicate that, in BRD2(2), a hierarchical mechanism of protein folding can be described with non-native interactions that play a significant role in folding.

Characterization of human frataxin missense variants in cancer tissues.

Human frataxin is an iron-binding protein involved in the mitochondrial iron-sulfur (Fe-S) clusters assembly, a process fundamental for the functional activity of mitochondrial proteins. Decreased level of frataxin expression is associated with the neurodegenerative disease Friedreich ataxia. Defective function of frataxin may cause defects in mitochondria, leading to increased tumorigenesis. Tumor-initiating cells show higher iron uptake, a decrease in iron storage and a reduced Fe-S clusters synthesis and utilization. In this study, we selected, from COSMIC database, the somatic human frataxin missense variants found in cancer tissues p.D104G, p.A107V, p.F109L, p.Y123S, p.S161I, p.W173C, p.S181F, and p.S202F to analyze the effect of the single amino acid substitutions on frataxin structure, function, and stability. The spectral properties, the thermodynamic and the kinetic stability, as well as the molecular dynamics of the frataxin missense variants found in cancer tissues point to local changes confined to the environment of the mutated residues. The global fold of the variants is not altered by the amino acid substitutions; however, some of the variants show a decreased stability and a decreased functional activity in comparison with that of the wild-type protein.

Evaluating the predictions of the protein stability change upon single amino acid substitutions for the FXN CAGI5 challenge.

Frataxin (FXN) is a highly conserved protein found in prokaryotes and eukaryotes that is required for efficient regulation of cellular iron homeostasis. Experimental evidence associates amino acid substitutions of the FXN to Friedreich Ataxia, a neurodegenerative disorder. Recently, new thermodynamic experiments have been performed to study the impact of somatic variations identified in cancer tissues on protein stability. The Critical Assessment of Genome Interpretation (CAGI) data provider at the University of Rome measured the unfolding free energy of a set of variants (FXN challenge data set) with far-UV circular dichroism and intrinsic fluorescence spectra. These values have been used to calculate the change in unfolding free energy between the variant and wild-type proteins at zero concentration of denaturant (ΔΔGH2O) . The FXN challenge data set, composed of eight amino acid substitutions, was used to evaluate the performance of the current computational methods for predicting the ΔΔGH2O value associated with the variants and to classify them as destabilizing and not destabilizing. For the fifth edition of CAGI, six independent research groups from Asia, Australia, Europe, and North America submitted 12 sets of predictions from different approaches. In this paper, we report the results of our assessment and discuss the limitations of the tested algorithms.

Single-Nucleotide Polymorphism of PPARγ, a Protein at the Crossroads of Physiological and Pathological Processes.

Genome polymorphisms are responsible for phenotypic differences between humans and for individual susceptibility to genetic diseases and therapeutic responses. Non-synonymous single-nucleotide polymorphisms (nsSNPs) lead to protein variants with a change in the amino acid sequence that may affect the structure and/or function of the protein and may be utilized as efficient structural and functional markers of association to complex diseases. This study is focused on nsSNP variants of the ligand binding domain of PPARγ a nuclear receptor in the superfamily of ligand inducible transcription factors that play an important role in regulating lipid metabolism and in several processes ranging from cellular differentiation and development to carcinogenesis. Here we selected nine nsSNPs variants of the PPARγ ligand binding domain, V290M, R357A, R397C, F360L, P467L, Q286P, R288H, E324K, and E460K, expressed in cancer tissues and/or associated with partial lipodystrophy and insulin resistance. The effects of a single amino acid change on the thermodynamic stability of PPARγ, its spectral properties, and molecular dynamics have been investigated. The nsSNPs PPARγ variants show alteration of dynamics and tertiary contacts that impair the correct reciprocal positioning of helices 3 and 12, crucially important for PPARγ functioning.

Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible for onset and maintenance of the cell transformation state, and they represent appropriate targets for development of diagnostic and therapeutic tools. METHODS: The unmutated E6 gene from HPV16 and HPV18 and from low-risk HPV11 was cloned in a prokaryotic expression vector for expression of the Histidine-tagged E6 protein (His6-E6), according to a novel procedure. The structural properties were determined using circular dichroism and fluorescence spectroscopy. His6-E6 oncoprotein immunogenicity was assessed in a mouse model, and its functionality was determined using in vitro GST pull-down and protein degradation assays. RESULTS: The His6-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes, without any further modification in the amino-acid sequence, were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His6-E6 suggests that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His6-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein-protein interactions. HR-HPV His6-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active. The purified HR-HPV E6 proteins also targeted the MAGI-3 and p53 proteins for degradation. CONCLUSIONS: This new procedure generates a stable, unmutated HPV16 E6 protein, which maintains the E6 properties in in vitro binding assays. This will be useful for basic studies, and for development of diagnostic kits and immunotherapies in preclinical mouse models of HPV-related tumorigenesis.

Computational and experimental studies on ß-sheet breakers targeting Aß1-40 fibrils.

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aß1-40 peptide in water in interaction with short peptides (ß-sheet breakers) mimicking the 17-21 region of the Aß1-40 sequence. Various systems differing in the customized ß-sheet breaker structure have been studied. Specifically we have considered three kinds of ß-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that ß-sheet breakers are able to inhibit in vitro fibril formation and prevent the ß sheet folding of portions of the Aß1-40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 ß-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aß1-40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aß1-40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17-21 Aß1-40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 ß-sheet breaker.

Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy.

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.

Mutation in the Common Docking Domain Affects MAP Kinase ERK2 Catalysis and Stability.

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.

Novel Arginine- and Proline-Rich Candidacidal Peptides Obtained through a Bioinformatic Approach.

Antimicrobial resistance is a major public health concern worldwide. Albeit to a lesser extent than bacteria, fungi are also becoming increasingly resistant to antifungal drugs. Moreover, due to the small number of antifungal classes, therapy options are limited, complicating the clinical management of mycoses. In this view, antimicrobial peptides (AMPs) are a potential alternative to conventional drugs. Among these, Proline-rich antimicrobial peptides (PrAMPs), almost exclusively of animal origins, are of particular interest due to their peculiar mode of action. In this study, a search for new arginine- and proline-rich peptides from plants has been carried out with a bioinformatic approach by sequence alignment and antimicrobial prediction tools. Two peptide candidates were tested against planktonic cells and biofilms of Candida albicans and Candida glabrata strains, including resistant isolates. These peptides showed similar potent activity, with half-maximal effective concentration values in the micromolar range. In addition, some structural and functional features, revealing peculiar mechanistic behaviors, were investigated.

Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many ß-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

Zn-Induced Interactions Between SARS-CoV-2 orf7a and BST2/Tetherin.

We present in this work a first X-ray Absorption Spectroscopy study of the interactions of Zn with human BST2/tetherin and SARS-CoV-2 orf7a proteins as well as with some of their complexes. The analysis of the XANES region of the measured spectra shows that Zn binds to BST2, as well as to orf7a, thus resulting in the formation of BST2-orf7a complexes. This structural information confirms the the conjecture, recently put forward by some of the present Authors, according to which the accessory orf7a (and possibly also orf8) viral protein are capable of interfering with the BST2 antiviral activity. Our explanation for this behavior is that, when BST2 gets in contact with Zn bound to the orf7a Cys15 ligand, it has the ability of displacing the metal owing to the creation of a new disulfide bridge across the two proteins. The formation of this BST2-orf7a complex destabilizes BST2 dimerization, thus impairing the antiviral activity of the latter.

Thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile Aeropyrum pernix.

The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.

Humanization of a highly stable single-chain antibody by structure-based antigen-binding site grafting.

The murine single-chain variable fragment F8 (scFv(F8)) is endowed with high intrinsic thermodynamic stability and can be functionally expressed in the reducing environment of both prokaryotic and eukaryotic cytoplasm. The stability and intracellular functionality of this molecule can be ascribed mostly to its framework regions and are essentially independent of the specific sequence and structure of the supported antigen-binding site. Therefore, the scFv(F8) represents a suitable scaffold to construct stable scFv chimeric molecules against different antigens by in vitro evolution or antigen-binding site grafting. Thanks to the favourable pharmacokinetic properties associated to a high thermodynamic stability of antibody fragments, such scFv(F8) variants may be exploited for a wide range of biomedical applications, from in vivo diagnosis to therapy, as well as to interfere with the function of intracellular proteins and pathogens, and for functional genomics studies. However, the potential immunogenicity of the murine framework regions represents a limitation for their exploitation in therapeutic applications. To overcome this limitation, we humanized a derivative of the scFv(F8), the anti-lysozyme scFv(11E), which is endowed with even higher thermodynamic stability than the parent antibody. The humanization was carried out by substituting the framework residues differing from closely related V(H) and V(L) domains of human origin with their human counterparts. Site-directed mutagenesis generated the fully humanized product and four intermediate scFvs, which were analyzed for protein expression and antigen binding. We found that the substitution Tyr 90-->Phe in the V(H) domain dramatically reduced the bacterial expression of all mutants. The back-mutation of Phe H90 to Tyr led to the final humanized variant named scFv(H5)H90Tyr. This molecule comprises humanized V(H) and V(L) framework regions and is endowed with HEL-binding affinity, stability in human serum and functionality under reducing conditions comparable to the murine cognate antibody. Consequently, the humanized scFv(H5)H90Tyr represents a suitable scaffold onto which new specificities towards antigens of therapeutic interest can be engineered for biomedical applications.

Dealing with Cu reduction in X-ray absorption spectroscopy experiments.

In this paper we prove in the exemplary case of the amyloid-ß peptide in complex with Cu(ii) that at the current low temperatures employed in XAS experiments, the time needed for collecting a good quality XAS spectrum is significantly shorter than the time after which structural damage becomes appreciable. Our method takes advantage of the well-known circumstance that the transition of Cu from the oxidized to the reduced form under ionizing radiation can be quantified by monitoring a characteristic peak in the pre-edge region. We show that there exists a sufficiently large time window in which good XAS spectra can be acquired before the structure around the oxidized Cu(ii) ion reorganizes itself into the reduced Cu(i) "resting" structure. We suggest that similar considerations apply to other cases of biological interest, especially when dealing with macromolecules in complex with transition metal ions.

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi.

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.

The phosphoglycerate kinase 1 variants found in carcinoma cells display different catalytic activity and conformational stability compared to the native enzyme.

Cancer cells are able to survive in difficult conditions, reprogramming their metabolism according to their requirements. Under hypoxic conditions they shift from oxidative phosphorylation to aerobic glycolysis, a behavior known as Warburg effect. In the last years, glycolytic enzymes have been identified as potential targets for alternative anticancer therapies. Recently, phosphoglycerate kinase 1 (PGK1), an ubiquitous enzyme expressed in all somatic cells that catalyzes the seventh step of glycolysis which consists of the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to ADP, has been discovered to be overexpressed in many cancer types. Moreover, several somatic variants of PGK1 have been identified in tumors. In this study we analyzed the effect of the single nucleotide variants found in cancer tissues on the PGK1 structure and function. Our results clearly show that the variants display a decreased catalytic efficiency and/or thermodynamic stability and an altered local tertiary structure, as shown by the solved X-ray structures. The changes in the catalytic properties and in the stability of the PGK1 variants, mainly due to the local changes evidenced by the X-ray structures, suggest also changes in the functional role of PGK to support the biosynthetic need of the growing and proliferating tumour cells.

The mutations Lys 114 --> Gln and Asp 126 --> Asn disrupt an intersubunit salt bridge and convert Listeria innocua Dps into its natural mutant Listeria monocytogenes Dps. Effects on protein stability at Low pH.

The stability of the dodecameric Listeria monocytogenes Dps has been compared with that of the Listeria innocua protein. The two proteins differ only in two amino acid residues that form an intersubunit salt-bridge in L. innocua Dps. This salt-bridge is replaced by a hydrogen bonding network in L. monocytogenes Dps as revealed by the X-ray crystal structure. The resistance to low pH and high temperature was assayed for both Dps proteins under equilibrium conditions and kinetically. Despite the identical equilibrium behavior, significant differences in the kinetic stability and activation energy of the unfolding process are apparent at pH 1.5. The higher stability of L. monocytogenes Dps has been accounted for in terms of the persistence of the hydrogen bonding network at this low pH value. In contrast, the salt-bridge between Lys 114 and Asp 126 characteristic of L. innocua Dps is most likely abolished due to protonation of Asp 126.