Catechol-induced covalent modifications modulate the aggregation tendency of α-synuclein: An in-solution and in-silico study.

Parkinson's disease (PD) stands as a challenging neurodegenerative condition characterized by the emergence of Lewy Bodies (LBs), intracellular inclusions within dopaminergic neurons. These LBs harbor various proteins, prominently including α-Synuclein (Syn) aggregates, implicated in disease pathology. A promising avenue in PD treatment involves targeting Syn aggregation. Recent findings from our research have shown that 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET) possess the ability to impede the formation of Syn fibrils by disrupting the aggregation process. Notably, these compounds primarily engage in noncovalent interactions with the protein, leading to the formation of off-pathway oligomers that deter fibril growth. Through proteolysis studies and mass spectrometry (MS) analysis, we have identified potential covalent modifications of Syn in the presence of DOPAC, although the exact site remains elusive. Employing molecular dynamics simulations, we delved into how DOPAC-induced covalent alterations might affect the mechanism of Syn aggregation. Our findings indicate that the addition of a covalent adduct on certain residues enhances fibril flexibility without compromising its secondary structure stability. Furthermore, in the monomeric state, the modified residue fosters novel bonding interactions, thereby influencing long-range interactions between the N- and C-termini of the protein.

RING 4.0: faster residue interaction networks with novel interaction types across over 35,000 different chemical structures.

Residue interaction networks (RINs) are a valuable approach for representing contacts in protein structures. RINs have been widely used in various research areas, including the analysis of mutation effects, domain-domain communication, catalytic activity, and molecular dynamics simulations. The RING server is a powerful tool to calculate non-covalent molecular interactions based on geometrical parameters, providing high-quality and reliable results. Here, we introduce RING 4.0, which includes significant enhancements for identifying both covalent and non-covalent bonds in protein structures. It now encompasses seven different interaction types, with the addition of π-hydrogen, halogen bonds and metal ion coordination sites. The definitions of all available bond types have also been refined and RING can now process the complete PDB chemical component dictionary (over 35000 different molecules) which provides atom names and covalent connectivity information for all known ligands. Optimization of the software has improved execution time by an order of magnitude. The RING web server has been redesigned to provide a more engaging and interactive user experience, incorporating new visualization tools. Users can now visualize all types of interactions simultaneously in the structure viewer and network component. The web server, including extensive help and tutorials, is available from URL: https://ring.biocomputingup.it/.

A PDZ scaffolding/CaM-mediated pathway in Cryptochrome signaling.

Cryptochromes are cardinal constituents of the circadian clock, which orchestrates daily physiological rhythms in living organisms. A growing body of evidence points to their participation in pathways that have not traditionally been associated with circadian clock regulation, implying that cryptochromes may be subject to modulation by multiple signaling mechanisms. In this study, we demonstrate that human CRY2 (hCRY2) forms a complex with the large, modular scaffolding protein known as Multi-PDZ Domain Protein 1 (MUPP1). This interaction is facilitated by the calcium-binding protein Calmodulin (CaM) in a calcium-dependent manner. Our findings suggest a novel cooperative mechanism for the regulation of mammalian cryptochromes, mediated by calcium ions (Ca2+ ) and CaM. We propose that this Ca2+ /CaM-mediated signaling pathway may be an evolutionarily conserved mechanism that has been maintained from Drosophila to mammals, most likely in relation to its potential role in the broader context of cryptochrome function and regulation. Further, the understanding of cryptochrome interactions with other proteins and signaling pathways could lead to a better definition of its role within the intricate network of molecular interactions that govern circadian rhythms.

Role of Helicobacter pylori Infection in Pathogenesis, Evolution, and Complication of Atherosclerotic Plaque.

The therapeutic management of atherosclerosis focuses almost exclusively on the reduction of plasma cholesterol levels. An important role in the genesis and evolution of atherosclerosis is played by chronic inflammation in promoting thrombosis phenomena after atheroma rupture. This review aims to take stock of the knowledge so far accumulated on the role of endemic HP infection in atherosclerosis. The studies produced so far have demonstrated a causal relationship between Helicobacter pylori (HP) and CVD. In a previous study, we demonstrated in HP-positive patients that thrombin and plasma fragment 1 + 2 production was proportionally related to tumor necrosis factor-alpha levels and that eradication of the infection resulted in a reduction of inflammation. At the end of our review, we can state that HP slightly affects the risk of CVD, particularly if the infection is associated with cytotoxic damage, and HP screening could have a clinically significant role in patients with a high risk of CVD. Considering the high prevalence of HP infection, an infection screening could be of great clinical utility in patients at high risk of CVD.

Vitamin D: Can Gender Medicine Have a Role?

This narrative review aims to shed light on the role of gender differences, on the biological and molecular functions in the main pathological mechanisms that recognize the role of vitamin D. Vitamin D deficiency is widespread worldwide, but it is still very controversial whether the amount of vitamin D taken daily is actually the only problem related to its biological functions. Currently, the plasma concentration of 25-hydroxyvitamin D represents the only indicator of the circulating blood quota. The concept is that the biological function of vitamin D is not only linked to its circulating levels, but it is hypothesized that its biological functions depend, above all, on its total bioavailability. In particular, vitamin D circulates for the most part linked to albumin and vitamin D binding protein (DBP), which depend on various pathological conditions and physiologically, above all, the function of the latter is regulated by estrogens, glucocorticoids, and inflammatory cytokines. During her life, women undergo various changes in the hormonal and sexual sphere concerning menarche, possible pregnancies, and breastfeeding but also the use of contraceptives and, finally, the transition from the period of fertility to menopause. Each of these phases presents specific needs and, consequently, sometimes also specific criticalities. Studies on young women have shown that vitamin D deficiency is present in 58 to 91% of cases. Obesity, metabolic disorders, and variation in estrogen contraction may affect vitamin D deficiency due to the decreased bioavailability from dietary sources due to deposition in body fat compartments.

Microbiota and Glucidic Metabolism: A Link with Multiple Aspects and Perspectives.

The global prevalence of overweight and obesity has dramatically increased in the last few decades, with a significant socioeconomic burden. In this narrative review, we include clinical studies aiming to provide the necessary knowledge on the role of the gut microbiota in the development of diabetic pathology and glucose-metabolism-related disorders. In particular, the role of a certain microbial composition of the fermentative type seems to emerge without a specific link to the development in certain subjects of obesity and the chronic inflammation of the adipose tissues, which underlies the pathological development of all the diseases related to glucose metabolism and metabolic syndrome. The gut microbiota plays an important role in glucose tolerance. Conclusion. New knowledge and new information is presented on the development of individualized therapies for patients affected by all the conditions related to reduced glucose tolerance and insulin resistance.

Cerebral Sinus Vein Thrombosis and Gender: A Not Entirely Casual Relationship.

Cerebral sinus venous thrombosis (CSVT) is a relatively rare acute disorder of cerebral circulation, but it can potentially be associated with serious sequelae and a poor prognosis. The neurological manifestations associated with it are often not adequately taken into consideration given the extreme variability and nuances of its clinical presentation and given the need for radiological methods suitable for this type of diagnosis. CSVT is usually more common in women, but so far there are little data available in the literature on sex-specific characteristics regarding this pathology. CSVT is the result of multiple conditions and is therefore to be considered a multifactorial disease where at least one risk factor is present in over 80% of cases. From the literature, we learn that congenital or acquired prothrombotic states are to be considered extremely associated with the occurrence of an acute episode of CSVT and its recurrences. It is, therefore, necessary to fully know the origins and natural history of CSVT, in order to implement the diagnostic and therapeutic pathways of these neurological manifestations. In this report, we summarize the main causes of CSVT considering the possible influence of gender, bearing in mind that most of the causes listed above are pathological conditions closely linked to the female sex.

RING-PyMOL: residue interaction networks of structural ensembles and molecular dynamics.

RING-PyMOL is a plugin for PyMOL providing a set of analysis tools for structural ensembles and molecular dynamic simulations. RING-PyMOL combines residue interaction networks, as provided by the RING software, with structural clustering to enhance the analysis and visualization of the conformational complexity. It combines precise calculation of non-covalent interactions with the power of PyMOL to manipulate and visualize protein structures. The plugin identifies and highlights correlating contacts and interaction patterns that can explain structural allostery, active sites, and structural heterogeneity connected with molecular function. It is easy to use and extremely fast, processing and rendering hundreds of models and long trajectories in seconds. RING-PyMOL generates a number of interactive plots and output files for use with external tools. The underlying RING software has been improved extensively. It is 10 times faster, can process mmCIF files and it identifies typed interactions also for nucleic acids. AVAILABILITY AND IMPLEMENTATION: https://github.com/BioComputingUP/ring-pymol.

Structural Characterization of Hypoxia Inducible Factor α-Prolyl Hydroxylase Domain 2 Interaction through MD Simulations.

The Prolyl Hydroxylases (PHDs) are an enzymatic family that regulates cell oxygen-sensing. PHDs hydroxylate hypoxia-inducible transcription factors α (HIFs-α) driving their proteasomal degradation. Hypoxia inhibits PHDs activity, inducing HIFs-α stabilization and cell adaptation to hypoxia. As a hallmark of cancer, hypoxia promotes neo-angiogenesis and cell proliferation. PHD isoforms are thought to have a variable impact on tumor progression. All isoforms hydroxylate HIF-α (HIF-1,2,3α) with different affinities. However, what determines these differences and how they pair with tumor growth is poorly understood. Here, molecular dynamics simulations were used to characterize the PHD2 binding properties in complexes with HIF-1α and HIF-2α. In parallel, conservation analysis and binding free energy calculations were performed to better understand PHD2 substrate affinity. Our data suggest a direct association between the PHD2 C-terminus and HIF-2α that is not observed in the PHD2/HIF-1α complex. Furthermore, our results indicate that phosphorylation of a PHD2 residue, Thr405, causes a variation in binding energy, despite the fact that this PTM has only a limited structural impact on PHD2/HIFs-α complexes. Collectively, our findings suggest that the PHD2 C-terminus may act as a molecular regulator of PHD's activity.

COVID-19 and Frailty.

Older age is a major risk factor for adverse outcomes of COVID-19, potentially due to immunosenescence and chronic low-grade inflammation, both characteristics of older adults which synergistically contribute to their vulnerability. Furthermore, older age is also associated with decreased kidney function and is consequently associated with an increased risk of cardiovascular disease. All of this in the course of COVID-19 infection can worsen and promote the progression of chronic kidney damage and all its sequelae. Frailty is a condition characterized by the decline in function of several homeostatic systems, leading to increased vulnerability to stressors and risk of adverse health outcomes. Thus, it is very likely that frailty, together with comorbidities, may have contributed to the high vulnerability to severe clinical manifestations and deaths from COVID-19 among older people. The combination of viral infection and chronic inflammation in the elderly could cause multiple unforeseen harmful consequences, affecting overall disability and mortality rates. In post-COVID-19 patients, inflammation has been implicated in sarcopenia progression, functional activity decline, and dementia. After the pandemic, it is imperative to shine a spotlight on these sequelae so that we can be prepared for the future outcomes of the ongoing pandemic. Here, we discuss the potential long-term consequences of SARS-CoV-2 infection and its possibility of causing permanent damage to the precarious balance existing in the frail elderly with multiple pathologies.

Antagonistic effect of cyclin-dependent kinases and a calcium-dependent phosphatase on polyglutamine-expanded androgen receptor toxic gain of function.

Spinal and bulbar muscular atrophy is caused by polyglutamine (polyQ) expansions in androgen receptor (AR), generating gain-of-function toxicity that may involve phosphorylation. Using cellular and animal models, we investigated what kinases and phosphatases target polyQ-expanded AR, whether polyQ expansions modify AR phosphorylation, and how this contributes to neurodegeneration. Mass spectrometry showed that polyQ expansions preserve native phosphorylation and increase phosphorylation at conserved sites controlling AR stability and transactivation. In small-molecule screening, we identified that CDC25/CDK2 signaling could enhance AR phosphorylation, and the calcium-sensitive phosphatase calcineurin had opposite effects. Pharmacologic and genetic manipulation of these kinases and phosphatases modified polyQ-expanded AR function and toxicity in cells, flies, and mice. Ablation of CDK2 reduced AR phosphorylation in the brainstem and restored expression of Myc and other genes involved in DNA damage, senescence, and apoptosis, indicating that the cell cycle-regulated kinase plays more than a bystander role in SBMA-vulnerable postmitotic cells.

Mutational Screening of Androgen Receptor Gene in 8224 Men of Infertile Couples.

CONTEXT: Mutations in the androgen receptor (AR) gene might be associated with infertility mainly because they cause various degrees of androgen insensitivity. OBJECTIVE: The aim of the study was to evaluate the frequency and type of AR variants in a large cohort of infertile males. METHODS: A total of 8224 males of Italian idiopathic infertile couples were referred to the University Hospital of Padova. The main outcome measures were mutational screening of AR, computational, and functional analyses. RESULTS: We found 131 patients (1.6%) harboring 45 variants in AR gene, of which 18 were novel missense AR variants. Patients with AR gene variants had lower sperm count (P = .048), higher testosterone (T) concentration (P < .0001), and higher androgen sensitivity index (ASI) (luteinizing hormone × T, P < .001) than patients without variants. Statistical analyses found T ≥ 15.38 nmol/L and ASI ≥ 180 IU × nmol/L2 as the threshold values to discriminate with good accuracy patients with AR variants. Patients with oligozoospermia and T ≥ 15.38 nmol/L had a 9-fold increased risk of harboring mutations compared with patients with normal sperm count and T < 15.38 nmol/L (odds ratio 9.29, 95% CI 5.07-17.02). Using computational and functional approaches, we identified 2 novel variants, L595P and L791I, as potentially pathogenic. CONCLUSION: This is the largest study screening AR gene variants in men of idiopathic infertile couples. We found that the prevalence of variants increased to 3.4% in oligozoospermic subjects with T ≥ 15.38 nmol/L. Conversely, more than 80% of men with AR gene variants had low sperm count and high T levels. Based on our findings, we suggest AR sequencing as a routine genetic test in cases of idiopathic oligozoospermia with T ≥ 15.38 nmol/L.

Investigating the Retained Inhibitory Effect of Cobimetinib against p.P124L Mutated MEK1: A Combined Liquid Biopsy and in Silico Approach.

The systemic treatment of metastatic melanoma has radically changed, due to an improvement in the understanding of its genetic landscape and the advent of targeted therapy. However, the response to BRAF/MEK inhibitors is transitory, and big efforts were made to identify the mechanisms underlying the resistance. We exploited a combined approach, encompassing liquid biopsy analysis and molecular dynamics simulation, for tracking tumor evolution, and in parallel defining the best treatment option. The samples at different time points were collected from a BRAF-mutant melanoma patient who developed an early resistance to dabrafenib/trametinib. The analysis of the circulating tumor DNA (ctDNA) identified the MEK1 p.P124L mutation that confers resistance to trametinib. With an in silico modeling, we identified cobimetinib as an alternative MEK inhibitor, and consequently suggested a therapy switch to vemurafenib/cobimetinib. The patient response was followed by ctDNA tracking and circulating melanoma cell (CMC) count. The cobimetinib administration led to an important reduction in the BRAF p.V600E and MEK1 p.P124L allele fractions and in the CMC number, features suggestive of a putative response. In summary, this study emphasizes the usefulness of a liquid biopsy-based approach combined with in silico simulation, to track real-time tumor evolution while assessing the best treatment option.

Mocafe: a comprehensive Python library for simulating cancer development with Phase Field Models.

SUMMARY: Mathematical models are effective in studying cancer development at different scales from metabolism to tissue. Phase Field Models (PFMs) have been shown to reproduce accurately cancer growth and other related phenomena, including expression of relevant molecules, extracellular matrix remodeling and angiogenesis. However, implementations of such models are rarely published, reducing access to these techniques. To reduce this gap, we developed Mocafe, a modular open-source Python package that implements some of the most important PFMs reported in the literature. Mocafe is designed to handle both PFMs purely based on differential equations and hybrid agent-based PFMs. Moreover, Mocafe is meant to be extensible, allowing the inclusion of new models in future releases. AVAILABILITY AND IMPLEMENTATION: Mocafe is a Python package based on FEniCS, a popular computing platform for solving partial differential equations. The source code, extensive documentation and demos are provided on GitHub at URL: https://github.com/BioComputingUP/mocafe. Moreover, we uploaded on Zenodo an archive of the package, which is available at https://doi.org/10.5281/zenodo.6366052.

Molecular Effects of Mutations in Human Genetic Diseases.

Next-generation sequencing (NGS) has enormously improved the identification of disease-candidate genetic variants [...].

RING 3.0: fast generation of probabilistic residue interaction networks from structural ensembles.

Residue interaction networks (RINs) are used to represent residue contacts in protein structures. Thanks to the advances in network theory, RINs have been proved effective as an alternative to coordinate data in the analysis of complex systems. The RING server calculates high quality and reliable non-covalent molecular interactions based on geometrical parameters. Here, we present the new RING 3.0 version extending the previous functionality in several ways. The underlying software library has been re-engineered to improve speed by an order of magnitude. RING now also supports the mmCIF format and provides typed interactions for the entire PDB chemical component dictionary, including nucleic acids. Moreover, RING now employs probabilistic graphs, where multiple conformations (e.g. NMR or molecular dynamics ensembles) are mapped as weighted edges, opening up new ways to analyze structural data. The web interface has been expanded to include a simultaneous view of the RIN alongside a structure viewer, with both synchronized and clickable. Contact evolution across models (or time) is displayed as a heatmap and can help in the discovery of correlating interaction patterns. The web server, together with an extensive help and tutorial, is available from URL: https://ring.biocomputingup.it/.

Expanding the clinical-pathological and genetic spectrum of RYR1-related congenital myopathies with cores and minicores: an Italian population study.

Mutations in the RYR1 gene, encoding ryanodine receptor 1 (RyR1), are a well-known cause of Central Core Disease (CCD) and Multi-minicore Disease (MmD). We screened a cohort of 153 patients carrying an histopathological diagnosis of core myopathy (cores and minicores) for RYR1 mutation. At least one RYR1 mutation was identified in 69 of them and these patients were further studied. Clinical and histopathological features were collected. Clinical phenotype was highly heterogeneous ranging from asymptomatic or paucisymptomatic hyperCKemia to severe muscle weakness and skeletal deformity with loss of ambulation. Sixty-eight RYR1 mutations, generally missense, were identified, of which 16 were novel. The combined analysis of the clinical presentation, disease progression and the structural bioinformatic analyses of RYR1 allowed to associate some phenotypes to mutations in specific domains. In addition, this study highlighted the structural bioinformatics potential in the prediction of the pathogenicity of RYR1 mutations. Further improvement in the comprehension of genotype-phenotype relationship of core myopathies can be expected in the next future: the actual lack of the human RyR1 crystal structure paired with the presence of large intrinsically disordered regions in RyR1, and the frequent presence of more than one RYR1 mutation in core myopathy patients, require designing novel investigation strategies to completely address RyR1 mutation effect.

SARS-CoV-2 variants preferentially emerge at intrinsically disordered protein sites helping immune evasion.

The SARS-CoV-2 pandemic is maintained by the emergence of successive variants, highlighting the flexibility of the protein sequences of the virus. We show that experimentally determined intrinsically disordered regions (IDRs) are abundant in the SARS-CoV-2 viral proteins, making up to 28% of disorder content for the S1 subunit of spike and up to 51% for the nucleoprotein, with the vast majority of mutations occurring in the 13 major variants mapped to these IDRs. Strikingly, antigenic sites are enriched in IDRs, in the receptor-binding domain (RBD) and in the N-terminal domain (NTD), suggesting a key role of structural flexibility in the antigenicity of the SARS-CoV-2 protein surface. Mutations occurring in the S1 subunit and nucleoprotein (N) IDRs are critical for immune evasion and antibody escape, suggesting potential additional implications for vaccines and monoclonal therapeutic strategies. Overall, this suggests the presence of variable regions on S1 and N protein surfaces, which confer sequence and antigenic flexibility to the virus without altering its protein functions.

Characterization of the pVHL Interactome in Human Testis Using High-Throughput Library Screening.

Functional impairment of the von Hippel-Lindau tumor suppressor (pVHL) is causative of a familiar increased risk of developing cancer. As an E3 substrate recognition particle, pVHL marks the hypoxia inducible factor 1α (HIF-1α) for degradation in normoxic conditions, thus acting as a key regulator of both acute and chronic cell adaptation to hypoxia. The male mice model carrying VHL gene conditional knockout presents significant abnormalities in testis development paired with defects in spermatogenesis and infertility, indicating that pVHL exerts testis-specific roles. Here we aimed to explore whether pVHL could have a similar role in humans by performing a testis-tissue library screening complemented with in-depth bioinformatics analysis. We identified 55 novel pVHL binding proteins directly involved in spermatogenesis, cell differentiation and reproductive metabolism. In addition, computational investigation of these new interactors identified multiple pVHL-specific binding motifs and demonstrated that somatic mutations described in human cancers reside in these binding regions. Collectively, these findings suggest that, in addition to its role in cancer formation, pVHL may also be pivotal in normal gonadal development in humans.

DisProt in 2022: improved quality and accessibility of protein intrinsic disorder annotation.

The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.