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.

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.

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.

The role of Zn ions in the interaction between SARS-CoV-2 orf7a protein and BST2/tetherin.

In this paper, we provide evidence that Zn ions play a role in the SARS-CoV-2 virus strategy to escape the immune response mediated by the BST2-tetherin host protein. This conclusion is based on sequence analysis and molecular dynamics simulations as well as X-ray absorption experiments [1].

Extracellular signal-Regulated Kinase 5 (ERK5) is required for the Yes-associated protein (YAP) co-transcriptional activity.

YES-associated protein (YAP) is a transcriptional cofactor with a key role in the regulation of several physio-pathological cellular processes, by integrating multiple cell autonomous and microenvironmental cues. YAP is the main downstream effector of the Hippo pathway, a tumor-suppressive signaling able to transduce several extracellular signals. The Hippo pathway acts restraining YAP activity, since its activation induces YAP phosphorylation and cytoplasmic sequestration.

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe, as it is customarily done, Co is most often used in experiments because Fe is extremely unstable owing to the fast oxidation reaction Fe → Fe. Protein stability is monitored following the conformational changes induced by Co binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements.

AMBRA1 regulates mitophagy by interacting with ATAD3A and promoting PINK1 stability.

PINK1 accumulation at the outer mitochondrial membrane (OMM) is a key event required to signal depolarized mitochondria to the autophagy machinery. How this early step is, in turn, modulated by autophagy proteins remains less characterized. Here, we show that, upon mitochondrial depolarization, the proautophagic protein AMBRA1 is recruited to the OMM and interacts with PINK1 and ATAD3A, a transmembrane protein that mediates mitochondrial import and degradation of PINK1.

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.

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.

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.

YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation.

Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified.

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

In this paper we prove in the exemplary case of the amyloid-β 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.

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.

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.

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.

Unveiling the folding mechanism of the Bromodomains.

Bromodomains (BRDs) are small protein domains often present in large multidomain proteins involved in transcriptional regulation in eukaryotic cells. They currently represent valuable targets for the development of inhibitors of aberrant transcriptional processes in a variety of human diseases. Here we report urea-induced equilibrium unfolding experiments monitored by circular dichroism (CD) and fluorescence on two structurally similar BRDs: BRD2(2) and BRD4(1), showing that BRD4(1) is more stable than BRD2(2).

The effect of β-sheet breaker peptides on metal associated Amyloid-β peptide aggregation process.

Far-UV Circular Dichroism experiments and Atomic Force Microscopy tomography are employed to assess the impact of β-sheet breakers on the Aβ peptide aggregation process in the presence of Cu or Zn transition metals. In this work we focus on two specific 5-amino acids long β-sheet breakers, namely the LPFFD Soto peptide, already known in the literature, and the LPFFN peptide recently designed and studied by our team. We provide evidence that both β-sheet breakers are effective in reducing the Aβ aggregation propensity, even in the presence of metal ions.

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.

PEGylated β-Sheet Breaker Peptides as Inhibitors of β-Amyloid Fibrillization.

Three PEGylated β-sheet breaker peptides are designed as new inhibitors of β-amyloid fibrillization. The peptide Ac-Leu-Pro-Phe-Phe-Asp-NH , considered the lead compound, and hexamers in which taurine and β-alanine substitute the acetyl group, are conjugated to poly(ethylene glycol); this conjugates self-assemble into nanoparticles. The activity of the PEGylated peptides as inhibitors of amyloid fibrillization are tested in vitro using circular dichroism spectroscopy and scanning electron microscopy.

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.

Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability.

Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors.

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.

Computational and experimental studies on β-sheet breakers targeting Aβ1-40 fibrils.

In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ1-40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17-21 region of the Aβ1-40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-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).

Molecular Characterization of the Multiple Interactions of SpsD, a Surface Protein from Staphylococcus pseudintermedius, with Host Extracellular Matrix Proteins.

Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes.