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Life sustains itself using energy generated by thermodynamic disequilibria, commonly existing as redox disequilibria. Metals are significant players in controlling redox reactions, as they are essential components of the engine that life uses to tap into the thermodynamic disequilibria necessary for metabolism. The number of proteins that evolved to catalyze redox reactions is extraordinary, as is the diversification level of metal cofactors and catalytic domain structures involved. Notwithstanding the importance of the topic, the relationship between metals and the redox reactions they are involved in has been poorly explored. This work reviews the structure and function of different prokaryotic organometallic-protein complexes, highlighting their pivotal role in controlling biogeochemistry. We focus on a specific subset of metal-containing oxidoreductases (EC1 or EC7.1), which are directly involved in biogeochemical cycles, i.e., at least one substrate or product is a small inorganic molecule that is or can be exchanged with the environment. Based on these inclusion criteria, we select and report 59 metalloenzymes, describing the organometallic structure of their active sites, the redox reactions in which they are involved, and their biogeochemical roles.

Fabry disease is caused by a deficiency of lysosomal alpha galactosidase and has a very large genotypic and phenotypic spectrum. Some patients who carry hypomorphic mutations can benefit from oral therapy with a pharmacological chaperone. The drug requires a very precise regimen because it is a reversible inhibitor of alpha-galactosidase. We looked for molecules that can potentiate this pharmacological chaperone, among drugs that have already been approved for other diseases. We tested candidate molecules in fibroblasts derived from a patient carrying a large deletion in the gene GLA, which were stably transfected with a plasmid expressing hypomorphic mutants. In our cell model, three drugs were able to potentiate the action of the pharmacological chaperone. We focused our attention on one of them, acetylsalicylic acid. We expect that acetylsalicylic acid can be used in synergy with the Fabry disease pharmacological chaperone and prolong its stabilizing effect on alpha-galactosidase.

The most common cause of human congenital disorders of glycosylation (CDG) are mutations in the phosphomannomutase gene PMM2, which affect protein N-linked glycosylation. The yeast gene SEC53 encodes a nearly-identical homolog of human PMM2. We evolved 384 populations of yeast harboring one of two human-disease-associated alleles, sec53-V238M and sec53-F126L, or wild-type SEC53. We find that after 1,000 generations, most populations compensate for the slow-growth phenotype associated with the sec53 human-disease-associated alleles. Through whole-genome sequencing we identify compensatory mutations, including known SEC53 genetic interactors. We observe an enrichment of compensatory mutations in other genes whose human homologs are associated with Type 1 CDG, including PGM1, which encodes the minor isoform of phosphoglucomutase in yeast. By genetic reconstruction, we show that evolved pgm1 mutations are dominant and allele-specific genetic interactors that restore both protein glycosylation and growth of yeast harboring the sec53-V238M allele. Finally, we characterize the enzymatic activity of purified Pgm1 mutant proteins. We find that reduction, but not elimination, of Pgm1 activity best compensates for the deleterious phenotypes associated with the sec53-V238M allele. Broadly, our results demonstrate the power of experimental evolution as a tool for identifying genes and pathways that compensate for human-disease associated alleles.

Observing mitral or tricuspid valve disease in an athlete raises many considerations for the clinician. Initially, the etiology must be clarified, with causes differing depending on whether the athlete is young or a master. Notably, vigorous training in competitive athletes leads to a constellation of structural and functional adaptations involving cardiac chambers and atrioventricular valve systems. In addition, a proper evaluation of the athlete with valve disease is necessary to evaluate the eligibility for competitive sports and identify those requiring more follow-up. Indeed, some valve pathologies are associated with an increased risk of severe arrhythmias and potentially sudden cardiac death. Traditional and advanced imaging modalities help clarify clinical doubts, allowing essential information about the athlete’s physiology and differentiating between primary valve diseases from those secondary to training-related cardiac adaptations. Remarkably, another application of multimodality imaging is evaluating athletes with valve diseases during exercise to reproduce the sport setting and better characterize the etiology and valve defect mechanism. This review aims to analyze the possible causes of atrioventricular valve diseases in athletes, focusing primarily on imaging applications in diagnosis and risk stratification.

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on β-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.

Enzyme replacement therapy is the only therapeutic option for Fabry patients with completely absent AGAL activity. However, the treatment has side effects, is costly, and requires conspicuous amounts of recombinant human protein (rh-AGAL). Thus, its optimization would benefit patients and welfare/health services (i.e., society at large). In this brief report, we describe preliminary results paving the way for two possible approaches: i. the combination of enzyme replacement therapy with pharmacological chaperones; and ii. the identification of AGAL interactors as possible therapeutic targets on which to act. We first showed that galactose, a low-affinity pharmacological chaperone, can prolong AGAL half-life in patient-derived cells treated with rh-AGAL. Then, we analyzed the interactomes of intracellular AGAL on patient-derived AGAL-defective fibroblasts treated with the two rh-AGALs approved for therapeutic purposes and compared the obtained interactomes to the one associated with endogenously produced AGAL (data available as PXD039168 on ProteomeXchange). Common interactors were aggregated and screened for sensitivity to known drugs. Such an interactor-drug list represents a starting point to deeply screen approved drugs and identify those that can affect (positively or negatively) enzyme replacement therapy.

Therapeutic solutions to counter

The term “pharmacological chaperone” was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.

The identification of high-risk factors for the infection by SARS-CoV-2 and the negative outcome of COVID-19 is crucial. The genetic background of the host might account for individual responses to SARS-CoV-2 infection besides age and comorbidities. A list of candidate polymorphisms is needed to drive targeted screens, given the existence of frequent polymorphisms in the general population. We carried out text mining in the scientific literature to draw up a list of genes referable to the term "SARS-CoV*". We looked for frequent mutations that are likely to affect protein function in these genes. Ten genes, mostly involved in innate immunity, and thirteen common variants were identified, for some of these the involvement in COVID-19 is supported by publicly available epidemiological data. We looked for available data on the population distribution of these variants and we demonstrated that the prevalence of five of them, Arg52Cys (rs5030737), Gly54Asp (rs1800450) and Gly57Glu (rs1800451) in MBL2, Ala59Thr (rs25680) in CD27, and Val197Met (rs12329760) in TMPRSS2, correlates with the number of cases and/or deaths of COVID-19 observed in different countries. The association of the TMPRSS2 variant provides epidemiological evidence of the usefulness of transmembrane protease serine 2 inhibitors for the cure of COVID-19. The identified genetic variants represent a basis for the design of a cost-effective assay for population screening of genetic risk factors in the COVID-19 pandemic.

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, &alpha, glucose-1,6-bisphosphate. The compound, &beta, glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. &beta, glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. &beta, glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.

The American FDA approved the first comprehensive NGS diagnostic assay for cancer at the end of 2017, leading the way to personalised therapy of cancer and the massive employ of bioinformatics (https://www.fda.gov/downloads/medicaldevices/productsandmedicalprocedures/invitrodiagnostics/ ucm584603.pdf ). In NGS-detected genes from cancer patients, driver and passenger mutations can be distinguished. The former provides a proliferative advantage to cancer cells and are commonly found, the latter do not provide proliferative fitness and are different in different patients. However, some passenger mutations might occur in genes involved in metabolism and could be mildly deleterious for cancer cells. Such deleteriousness could be increased using a specific inhibitor of the mutated protein product. A personalized therapy of cancer could address both driver and passenger mutations. To evaluate to which extent it is possible to address passenger mutations for the cure of cancers, we built a gene/ inhibitor list, crossing DrugBank, a database that combines detailed drug data with comprehensive drug target information, with COSMIC, the catalogue of somatic mutations in cancer. First, we obtained the approved drugs annotated as inhibitors from DrugBank, and the genes encoding their target proteins. We then looked for these genes in COSMIC, to check how many missense mutations have been detected in cancer patient genomes.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases caused by defects in glycosylation. Nearly 100 CDG types are known so far. Patients present a great phenotypic diversity ranging from poly- to mono-organ/system involvement and from very mild to extremely severe presentation. In this literature review, we summarize the liver involvement reported in CDG patients. Although liver involvement is present in only a minority of the reported CDG types (22 %), it can be debilitating or even life-threatening. Sixteen of the patients we collated here developed cirrhosis, 10 had liver failure. We distinguish two main groups: on the one hand, the CDG types with predominant or isolated liver involvement including MPI-CDG, TMEM199-CDG, CCDC115-CDG, and ATP6AP1-CDG, and on the other hand, the CDG types associated with liver disease but not as a striking, unique or predominant feature, including PMM2-CDG, ALG1-CDG, ALG3-CDG, ALG6-CDG, ALG8-CDG, ALG9-CDG, PGM1-CDG, and COG-CDG. This review aims to facilitate CDG patient identification and to understand CDG liver involvement, hopefully leading to earlier diagnosis, and better management and treatment.

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases comprising more than 85 known distinct disorders. They show a great phenotypic variability ranging from multi-organ/system to mono-organ/system involvement with very mild to extremely severe expression. Immunological dysfunction has a significant impact on the phenotype in a minority of CDG. CDG with major immunological involvement are ALG12-CDG, MAGT1-CDG, MOGS-CDG, SLC35C1-CDG and PGM3-CDG. This review discusses the variety of immunological abnormalities reported in human CDG. Understanding the immunological aspects of CDG may contribute to a better management/treatment of these pathologies and possibly of more common diseases, such as inflammatory diseases.

Thermal shift assay can be used for the high-throughput screening of pharmacological chaperones. These drugs are small molecules that bind a mutant protein and stabilize it. We demonstrated the robustness, reproducibility and versatility of the method using two molecules that are in clinical trial for Fabry or Pompe disease, Deoxygalactonojirimycin and N-Butyldeoxynojirimycin, and their target enzymes, lysosomal alpha-galactosidaseA and alpha-glucosidase, as test cases. We assessed the influence of solvents and of scanning rate on the measures. We showed that a value that is equivalent to the melting temperature can be obtained by the first derivatives of raw data. We discuss the advantages of the method and the precaution to be taken in running the experiments. © 2015 The Authors Drug Testing and Analysis Published by John Wiley & Sons Ltd.

Background: Rare diseases represent a challenge for physicians because patients are rarely seen, and they can manifest with symptoms similar to those of common diseases. In this work, genetic confirmation of diagnosis is derived from DNA sequencing. We present a tutorial for the molecular analysis of a rare disease using Fabry disease as an example. Methods: An exonic sequence derived from a hypothetical male patient was matched against human reference data using a genome browser. The missense mutation was identified by running BlastX, and information on the affected protein was retrieved from the database UniProt. The pathogenic nature of the mutation was assessed with PolyPhen-2. Disease-specific databases were used to assess whether the missense mutation led to a severe phenotype, and whether pharmacological therapy was an option. Results: An inexpensive bioinformatics approach is presented to get the reader acquainted with the diagnosis of Fabry disease. The reader is introduced to the field of pharmacological chaperones, a therapeutic approach that can be applied only to certain Fabry genotypes. Conclusion: The principle underlying the analysis of exome sequencing can be explained in simple terms using web applications and databases which facilitate diagnosis and therapeutic choices.