Biophysical characterization of melanoma cell phenotype markers during metastatic progression

Melanoma is the most fatal form of skin cancer, with increasing prevalence worldwide. The most common melanoma genetic driver is mutation of the proto-oncogene serine/threonine kinase BRAF; thus, the inhibition of its MAP kinase pathway by specific inhibitors is a commonly applied therapy. However, many patients are resistant, or develop resistance to this type of monotherapy, and therefore combined therapies which target other signaling pathways through various molecular mechanisms are required. A possible strategy may involve targeting cellular energy metabolism, which has been recognized as crucial for cancer development and progression and which connects through glycolysis to cell surface glycan biosynthetic pathways. Protein glycosylation is a hallmark of more than 50% of the human proteome and it has been recognized that altered glycosylation occurs during the metastatic progression of melanoma cells which, in turn facilitates their migration. This review provides a description of recent advances in the search for factors able to remodel cell metabolism between glycolysis and oxidative phosphorylation, and of changes in specific markers and in the biophysical properties of cells during melanoma development from a nevus to metastasis. This development is accompanied by changes in the expression of surface glycans, with corresponding changes in ligand-receptor affinity, giving rise to structural features and viscoelastic parameters particularly well suited to study by label-free biophysical methods

Human coronary inflammation by computed tomography: Relationship with coronary microvascular dysfunction

Background A new imaging metric using coronary computed tomography angiography (CCTA), addressing the peri-coronary adipose tissue (PCAT) computed tomography (CT) attenuation, has been clinically validated. This method provides information regarding coronary inflammation. It is unclear how coronary inflammation affects microvascular function. The non-invasive evaluation of coronary flow velocity reserve is widely used in clinical practice using Doppler measurement on the left anterior descending coronary artery (CFVR-lad) during stress-echocardiography (SE). We hypothesize that coronary inflammation affects CFVR-lad and, in the absence of overt CAD, they are significantly correlated. Methods We evaluated the relationship between coronary inflammation (by PCAT CT attenuation) and coronary microvascular function (by CFVR-lad) in subjects with no or non-obstructive (diameter stenosis <70%) coronary artery disease (CAD). Results Two-hundred and two subjects were enrolled in the study. The relationship between PCAT CT attenuation and CFVR-lad show a significant inverse relationship in the entire group of subjects enrolled in the study (r = −0.32, p < 0.001). Correlation between PCAT CT attenuation and CFVR-lad was significant in subjects with no or mild CAD-lad, while this was not the case in subjects with intermediate CAD-lad. The R and R2 were respectively −0.40, −0.16 in subjects without CAD (p < 0.001) and − 0.35 and − 0.12 in subjects with mild CAD-lad (p = 0.001). Conclusions The main finding of the current study is the independent relationship between coronary microvascular function, by Doppler CFVR-lad during SE, in subjects without severely obstructive CAD in the left anterior descending coronary artery, and the level of local coronary inflammation, by PCAT attenuation measurement on CCTA

Studying ggdef domain in the act: Minimize conformational frustration to prevent artefacts

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies

Targeting the Interaction between the SH3 Domain of Grb2 and Gab2

Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism

Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5'untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1-UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism

Clinical use of a 180-day implantable glucose sensor improves glycated haemoglobin and time in range in patients with type 1 diabetes

Aims: This real-world study evaluated the changes in glycated haemoglobin (HbA1c) and continuous glucose monitoring (CGM) metrics associated with use of the implantable 180-day Eversense CGM System (Eversense) in patients with type 1 diabetes. Materials and methods: This was a prospective, multicentre, observational study among adult participants aged ≥18 years with type 1 diabetes across seven diabetes-care centres in Italy who had Eversense inserted for the first time. HbA1c was measured at baseline and at 180 days. Changes in time in range [TIR (glucose 70–180 mg/dL)], time above range [TAR (glucose >180 mg/dL)], time below range [TBR (glucose <70 mg/dL)] and glycaemic variability were also assessed. Data were also analysed by previous CGM use and by mode of insulin delivery. Results: One-hundred patients were enrolled (mean age 36 ± 12 years, mean baseline HbA1c 7.4 ± 0.92% [57 ± 10 mmol/mol]). Fifty-six per cent of patients were users of the continuous subcutaneous insulin infusion pump and 45% were previous users of CGM. HbA1c significantly decreased in patients after 180 days of sensor wear (−0.43% ± 0.69%, 5 ± 8 mmol/mol, P < 0.0001). As expected, CGM-naïve patients achieved the greatest reduction in HbA1c (−0.74% ± 0.48%, 8 ± 5 mmol/mol). TIR significantly increased and TAR and mean daily sensor glucose significantly decreased while TBR did not change after 180 days of sensor wear. Conclusions: Real-world clinical use of the Eversense CGM System for 180 days was associated with significant improvements in HbA1c and CGM metrics among adults with type 1 diabetes. The study is registered on clinicaltrials.gov (NCT04160156)

High-fat diet leads to reduced protein o-glcnacylation and mitochondrial defects promoting the development of alzheimer\u2019s disease signatures

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer\u2019s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process

Retinoic acid-induced 1 gene haploinsufficiency alters lipid metabolism and causes autophagy defects in Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment

Structure and function of HD-GYP phosphodiesterases

HD-GYPs represent the least abundant, and somewhat mysterious, class of dedicated cyclic di-GMP phosphodiesterases (PDE). They are metal dependent enzymes, belonging to the HD phosphohydrolase superfamily, and are evolutionarily unrelated to the EAL class of cyclic di-GMP dedicated PDEs. In contrast to the EAL domain that hydrolyses cyclic di-GMP to pGpG, HD-GYPs are able to further hydrolyse pGpG to GMP. As both the GGDEF and EAL domains, the HD-GYP module is often found fused with other regulatory domains. Despite the ability to act as a PDE, the physiological role(s) of HD-GYP proteins within the cyclic di-GMPdependent biofilm regulation are still not fully clarified. Indeed, many HD-GYPs may also mediate protein protein interactions within more complex regulatory pathways or function as cyclic di-GMP or pGpG receptors. The few structures available indicate that HD-GYPs can be clustered into two distinct groups depending on the metal binding site, which can accommodate two or three metal ions. The nature and the number of bound metals determine whether a certain HD-GYP will be active as a PDE or will function as a dinucleotide binding domain. In this chapter, we will review the biochemical and structural data available to date on HD-GYPs