An Update on the Role of Ubiquitination in Melanoma Development and Therapies.

The ubiquitination system plays a critical role in regulation of large array of biological processes and its alteration has been involved in the pathogenesis of cancers, among them cutaneous melanoma, which is responsible for the most deaths from skin cancers. Over the last decades, targeted therapies and immunotherapies became the standard therapeutic strategies for advanced melanomas. However, despite these breakthroughs, the prognosis of metastatic melanoma patients remains unoptimistic, mainly due to intrinsic or acquired resistances. Many avenues of research have been investigated to find new therapeutic targets for improving patient outcomes. Because of the pleiotropic functions of ubiquitination, and because each step of ubiquitination is amenable to pharmacological targeting, much attention has been paid to the role of this process in melanoma development and resistance to therapies. In this review, we summarize the latest data on ubiquitination and discuss the possible impacts on melanoma treatments.

FBXO32 links ubiquitination to epigenetic reprograming of melanoma cells.

Ubiquitination by serving as a major degradation signal of proteins, but also by controlling protein functioning and localization, plays critical roles in most key cellular processes. Here, we show that MITF, the master transcription factor in melanocytes, controls ubiquitination in melanoma cells. We identified FBXO32, a component of the SCF E3 ligase complex as a new MITF target gene. FBXO32 favors melanoma cell migration, proliferation, and tumor development in vivo. Transcriptomic analysis shows that FBXO32 knockdown induces a global change in melanoma gene expression profile. These include the inhibition of CDK6 in agreement with an inhibition of cell proliferation and invasion upon FBXO32 silencing. Furthermore, proteomic analysis identifies SMARC4, a component of the chromatin remodeling complexes BAF/PBAF, as a FBXO32 partner. FBXO32 and SMARCA4 co-localize at loci regulated by FBXO32, such as CDK6 suggesting that FBXO32 controls transcription through the regulation of chromatin remodeling complex activity. FBXO32 and SMARCA4 are the components of a molecular cascade, linking MITF to epigenetics, in melanoma cells.

Osteogenic and Chondrogenic Master Genes Expression Is Dependent on the Kir2.1 Potassium Channel Through the Bone Morphogenetic Protein Pathway.

Andersen's syndrome is a rare disorder affecting muscle, heart, and bone that is associated with mutations leading to a loss of function of the inwardly rectifying K+ channel Kir2.1. Although the Kir2.1 function can be anticipated in excitable cells by controlling the electrical activity, its role in non-excitable cells remains to be investigated. Using Andersen's syndrome-induced pluripotent stem cells, we investigated the cellular and molecular events during the osteoblastic and chondrogenic differentiation that are affected by the loss of the Ik1 current. We show that loss of Kir2.1 channel function impairs both osteoblastic and chondrogenic processes through the downregulation of master gene expression. This downregulation is the result of an impairment of the bone morphogenetic proteins signaling pathway through dephosphorylation of the Smad proteins. Restoring Kir2.1 channel function in Andersen's syndrome cells rescued master genes expression and restored normal osteoblast and chondrocyte behavior. Our results show that Kir2.1-mediated activity controls endochondral and intramembranous ossification signaling pathways. © 2018 American Society for Bone and Mineral Research.

Substitutions of the S4DIV R2 residue (R1451) in NaV1.4 lead to complex forms of paramyotonia congenita and periodic paralyses.

Mutations in NaV1.4, the skeletal muscle voltage-gated Na+ channel, underlie several skeletal muscle channelopathies. We report here the functional characterization of two substitutions targeting the R1451 residue and resulting in 3 distinct clinical phenotypes. The R1451L is a novel pathogenic substitution found in two unrelated individuals. The first individual was diagnosed with non-dystrophic myotonia, whereas the second suffered from an unusual phenotype combining hyperkalemic and hypokalemic episodes of periodic paralysis (PP). The R1451C substitution was found in one individual with a single attack of hypoPP induced by glucocorticoids. To elucidate the biophysical mechanism underlying the phenotypes, we used the patch-clamp technique to study tsA201 cells expressing WT or R1451C/L channels. Our results showed that both substitutions shifted the inactivation to hyperpolarized potentials, slowed the kinetics of inactivation, slowed the recovery from slow inactivation and reduced the current density. Cooling further enhanced these abnormalities. Homology modeling revealed a disruption of hydrogen bonds in the voltage sensor domain caused by R1451C/L. We concluded that the altered biophysical properties of R1451C/L well account for the PMC-hyperPP cluster and that additional factors likely play a critical role in the inter-individual differences of clinical expression resulting from R1451C/L.

Andersen's syndrome mutants produce a knockdown of inwardly rectifying K+ channel in mouse skeletal muscle in vivo.

Andersen's syndrome (AS) is a rare autosomal disorder that has been defined by the triad of periodic paralysis, cardiac arrhythmia, and developmental anomalies. AS has been directly linked to over 40 different autosomal dominant negative loss-of-function mutations in the KCNJ2 gene, encoding for the tetrameric strong inward rectifying K+ channel KIR2.1. While KIR2.1 channels have been suggested to contribute to setting the resting membrane potential (RMP) and to control the duration of the action potential (AP) in skeletal and cardiac muscle, the mechanism by which AS mutations produce such complex pathophysiological symptoms is poorly understood. Thus, we use an adenoviral transduction strategy to study in vivo subcellular distribution of wild-type (WT) and AS-associated mutant KIR2.1 channels in mouse skeletal muscle. We determined that WT and D71V AS mutant KIR2.1 channels are localized to the sarcolemma and the transverse tubules (T-tubules) of skeletal muscle fibers, while the ∆314-315 AS KIR2.1 mutation prevents proper trafficking of the homo- or hetero-meric channel complexes. Whole-cell voltage-clamp recordings in individual skeletal muscle fibers confirmed the reduction of inwardly rectifying K+ current (IK1) after transduction with ∆314-315 KIR2.1 as compared to WT channels. Analysis of skeletal muscle function revealed reduced force generation during isometric contraction as well as reduced resistance to muscle fatigue in extensor digitorum longus muscles transduced with AS mutant KIR2.1. Together, these results suggest that KIR2.1 channels may be involved in the excitation-contraction coupling process required for proper skeletal muscle function. Our findings provide clues to mechanisms associated with periodic paralysis in AS.

A recessive Nav1.4 mutation underlies congenital myasthenic syndrome with periodic paralysis.

OBJECTIVE: To determine the molecular basis of a complex phenotype of congenital muscle weakness observed in an isolated but consanguineous patient. METHODS: The proband was evaluated clinically and neurophysiologically over a period of 15 years. Genetic testing of candidate genes was performed. Functional characterization of the candidate mutation was done in mammalian cell background using whole cell patch clamp technique. RESULTS: The proband had fatigable muscle weakness characteristic of congenital myasthenic syndrome with acute and reversible attacks of most severe muscle weakness as observed in periodic paralysis. We identified a novel homozygous SCN4A mutation (p.R1454W) linked to this recessively inherited phenotype. The p.R1454W substitution induced an important enhancement of fast and slow inactivation, a slower recovery for these inactivated states, and a frequency-dependent regulation of Nav1.4 channels in the heterologous expression system. CONCLUSION: We identified a novel loss-of-function mutation of Nav1.4 that leads to a recessive phenotype combining clinical symptoms and signs of congenital myasthenic syndrome and periodic paralysis, probably by decreasing channel availability for muscle action potential genesis at the neuromuscular junction and propagation along the sarcolemma.

The inward rectifier potassium channel Kir2.1 is required for osteoblastogenesis.

Andersen's syndrome (AS) is a rare and dominantly inherited pathology, linked to the inwardly rectifying potassium channel Kir2.1. AS patients exhibit a triad of symptoms that include periodic paralysis, cardiac dysrhythmia and bone malformations. Some progress has been made in understanding the contribution of the Kir2.1 channel to skeletal and cardiac muscle dysfunctions, but its role in bone morphogenesis remains unclear. We isolated myoblast precursors from muscle biopsies of healthy individuals and typical AS patients with dysmorphic features. Myoblast cultures underwent osteogenic differentiation that led to extracellular matrix mineralization. Osteoblastogenesis was monitored through the activity of alkaline phosphatase, and through the hydroxyapatite formation using Alizarin Red and Von Kossa staining techniques. Patch-clamp recordings revealed the presence of an inwardly rectifying current in healthy cells that was absent in AS osteoblasts, showing the dominant-negative effect of the Kir2.1 mutant allele in osteoblasts. We also found that while control cells actively synthesize hydroxyapatite, AS osteoblasts are unable to efficiently form any extracellular matrix. To further demonstrate the role of the Kir2.1 channels during the osteogenesis, we inhibited Kir2.1 channel activity in healthy patient cells by applying extracellular Ba(2+) or using adenoviruses carrying mutant Kir2.1 channels. In both cases, cells were no longer able to produce extracellular matrixes. Moreover, osteogenic activity of AS osteoblasts was restored by rescue experiments, via wild-type Kir2.1 channel overexpression. These observations provide a proof that normal Kir2.1 channel function is essential during osteoblastogenesis.

Proteomic analysis of lymphoblastoid cells from Nasu-Hakola patients: a step forward in our understanding of this neurodegenerative disorder.

Nasu-Hakola disease (NHD) is a recessively inherited rare disorder characterized by a combination of neuropsychiatric and bone symptoms which, while being unique to this disease, do not provide a rationale for the unambiguous identification of patients. These individuals, in fact, are likely to go unrecognized either because they are considered to be affected by other kinds of dementia or by fibrous dysplasia of bone. Given that dementia in NHD has much in common with Alzheimer's disease and other neurodegenerative disorders, it cannot be expected to achieve the differential diagnosis of this disease without performing a genetic analysis. Under this scenario, the availability of protein biomarkers would indeed provide a novel context to facilitate interpretation of symptoms and to make the precise identification of this disease possible. The work here reported was designed to generate, for the first time, protein profiles of lymphoblastoid cells from NHD patients. Two-dimensional electrophoresis (2-DE) and nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) have been applied to all components of an Italian family (seven subjects) and to five healthy subjects included as controls. Comparative analyses revealed differences in the expression profile of 21 proteins involved in glucose metabolism and information pathways as well as in stress responses.

Respiratory Proteomics Today: Are Technological Advances for the Identification of Biomarker Signatures Catching up with Their Promise? A Critical Review of the Literature in the Decade 2004-2013.

To improve the knowledge on a variety of severe disorders, research has moved from the analysis of individual proteins to the investigation of all proteins expressed by a tissue/organism. This global proteomic approach could prove very useful: (i) for investigating the biochemical pathways involved in disease; (ii) for generating hypotheses; or (iii) as a tool for the identification of proteins differentially expressed in response to the disease state. Proteomics has not been used yet in the field of respiratory research as extensively as in other fields, only a few reproducible and clinically applicable molecular markers, which can assist in diagnosis, having been currently identified. The continuous advances in both instrumentation and methodology, which enable sensitive and quantitative proteomic analyses in much smaller amounts of biological material than before, will hopefully promote the identification of new candidate biomarkers in this area. The aim of this report is to critically review the application over the decade 2004-2013 of very sophisticated technologies to the study of respiratory disorders. The observed changes in protein expression profiles from tissues/fluids of patients affected by pulmonary disorders opens the route for the identification of novel pathological mediators of these disorders.

An insight into the abundant proteome of 46BR.1G1 fibroblasts deficient of DNA ligase I.

This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.

Phosphorylation of SRSF1 is modulated by replicational stress.

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.

Recent novel MEKC applications to analyze free amino acids in different biomatrices: 2009-2010.

This report intends to provide an updated overview of the most important methodological developments of MEKC in the field of qualitative/quantitative analysis of free amino acids in different matrices. A good number of articles published in the years 2009-2010 addresses the main applications of such procedures together with their advantages and/or drawbacks. The usefulness of chiral CE selective methods for the separation of D-amino acids in biological and food samples and the use of microchips, as well as other foreseen trends in different areas, are also discussed.

Indoleamine 2,3-dioxygenase in lung allograft tolerance.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the degradation of tryptophan (Try) to kynurenine (Kyn), is thought to suppress T-cell activity. Although a few experimental studies have suggested a role for IDO in graft acceptance, human data are scarce and inconclusive. We sought to establish whether, in lung transplant recipients (LTRs), plasma IDO activity mirrors the level of graft acceptance. METHODS: We measured the plasma Kyn/Try ratio, reflecting IDO activity, by high-performance liquid chromatography (HPLC) in 90 LTRs, including 26 patients who were still functionally/clinically stable for >36 post-transplant months (stable LTRs) and 64 LTRs with bronchiolitis obliterans syndrome (BOS, Grades 0-p to 3). Twenty-four normal healthy controls (NHCs) were also included. RESULTS: The Kyn/Try ratio in stable LTRs resembled that observed in NHCs, whereas, unexpectedly, patients with BOS, who had lower counts of peripheral CD4(+) T-regulatory cells and tolerogenic plasmacytoid dendritic cells than stable LTRs, showed an increased plasma Kyn/Try ratio compared with both NHCs and stable LTRs. IDO expression by in vitro-stimulated peripheral blood mononuclear cells (PBMC) did not vary between BOS and stable LTRs. Furthermore, BOS patients displayed signs of chronic systemic inflammation (increased plasma levels of interleukin-8 and tumor necrosis factor-alpha) and higher T-cell activation (increased frequency of peripheral interferon-gamma-producing clones). CONCLUSIONS: Our results suggest that, in vivo, in lung transplantation, plasma IDO activity does not reflect the degree of lung graft acceptance, but instead is correlated with the degree of chronic inflammation.

2-DE and LC-MS/MS for a comparative proteomic analysis of BALf from subjects with different subsets of inflammatory myopathies.

The protein profiles of bronchoalveolar lavage fluid (BALf) of patients belonging to three selected subsets of Polymyositis/Dermatomyositis (PM/DM) have been compared by using a combination of 2-DE and MALDI-TOF/MS or LC-MS/MS. Our study examined the hypothesis that there were distinct differences in protein expression profiles that were related to the phenotype. From among the 323+/-51 protein spots that may represent the most highly expressed proteins in BALf of these patients, 24 unique spots were isolated and proteins identified. In particular, 9 spots were present in BALf of PM/DM patients only; 12 spots were exclusive of Overlap patients and 3 spots of AS patients. From among the proteins identified, a few were classified as cytoskeletal proteins, others were involved in oxidative stress and a number of proteins were associated with general metabolic activity or immunological response and inflammation. This is the first study in which evidence is provided that a number of different proteins are expressed in different subsets of PM/DM and supports our contention that the proteomic approach would be beneficial in discovering molecules which could represent possible prognostic factors of these rare pathologies.