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Huntington's disease (HD) is a lethal neurodegenerative disorder for which no cure is available yet. It is caused by abnormal expansion of a CAG triplet in the gene encoding the huntingtin protein (Htt), with consequent expansion of a polyglutamine repeat in mutated Htt (mHtt). This makes mHtt highly unstable and aggregation prone. Soluble mHtt is linked to cytotoxicity and neurotoxicity, whereas mHtt aggregates are thought to be neuroprotective. While Htt and mHtt are ubiquitously expressed throughout the brain and peripheral tissues, HD is characterized by selective degradation of the corpus striatum, without notable alterations in peripheral tissues. Screening for mRNAs preferentially expressed in rodent striatum led to the discovery of a GTP binding protein homologous to Ras family members. Due to these features, the newly discovered protein was termed Ras Homolog Enriched in Striatum (RHES). The aetiological role of RHES in HD has been ascribed to its small ubiquitin-like modifier (SUMO)-E3 ligase function. RHES sumoylates mHtt with higher efficiency than wild-type Htt, thereby protecting mHtt from degradation and increasing the amounts of the soluble form. Although RHES is an attractive target for HD treatment, essential information about protein structure and function are still missing. With the aim of investigating RHES 3D structure and function, bioinformatic analyses and molecular modelling have been performed in the present study, based on which, RHES regions predicted to be involved in the interaction with mHtt or the SUMO-E2 ligase Ubc9 have been identified. These regions have been used to design peptides aimed at inhibiting RHES interactions and, therefore, mHtt sumoylation; in turn, these peptides will be used to develop small molecule inhibitors by both rational design and virtual screening of large compound libraries. Once identified, RHES sumoylation inhibitors may open the road to the development of therapeutic agents against the severe, and currently untreatable, HD.

Vascular endothelial growth factor receptor (VEGFR)-1 exists in different forms, derived from alternative splicing of the same gene. In addition to the transmembrane form, endothelial cells produce a soluble VEGFR-1 (sVEGFR-1) isoform, whereas non-endothelial cells produce both sVEGFR-1 and a different soluble molecule, known as soluble fms-like tyrosine kinase (sFlt)1-14. By binding members of the vascular endothelial growth factor (VEGF) family, the soluble forms reduce the amounts of VEGFs available for the interaction with their transmembrane receptors, thereby negatively regulating VEGFR-mediated signaling. In agreement with this activity, high levels of circulating sVEGFR-1 or sFlt1-14 are associated with different pathological conditions involving vascular dysfunction. Moreover, sVEGFR-1 and sFlt1-14 have an additional role in angiogenesis: they are deposited in the endothelial cell and pericyte extracellular matrix, and interact with cell membrane components. Interaction of sVEGFR-1 with α5β1 integrin on endothelial cell membranes regulates vessel growth, triggering a dynamic, pro-angiogenic phenotype. Interaction of sVEGFR-1/sFlt1-14 with cell membrane glycosphingolipids in lipid rafts controls kidney cell morphology and glomerular barrier functions. These cell–matrix contacts represent attractive novel targets for pharmacological intervention in addition to those addressing interactions between VEGFs and their receptors.

Magnetic nanoparticles, MNPs, mineralized within a human ferritin protein cage, HFt, can represent an appealing platform to realize smart therapeutic agents for cancer treatment by drug delivery and magnetic fluid hyperthermia, MFH. However, the constraint imposed by the inner diameter of the protein shell (ca. 8 nm) prevents its use as heat mediator In MFH when the MNPs comprise pure iron oxide. In this contribution, we demonstrate how this limitation can be overcome through the controlled doping of the core with small amount of Co(II). Highly monodisperse doped iron oxide NPs with average size of 7 nm are mineralized inside a genetically modified variant of HFt, carrying several copies of a-melanocyte-stimulating hormone peptide, which has already been demonstrated to have excellent targeting properties toward melanoma cells. HFt is also conjugated to poly(ethylene glycol) molecules to increase its in vivo stability. The investigation of hyperthermic properties of HFt-NPs shows that a Co doping of 5% Is enough to strongly enhance the magnetic anisotropy and thus the hyperthermic efficiency with respect to the undoped sample. In vitro tests performed on B16 melanoma cell line demonstrate a strong reduction of the cell viability after treatment with Co doped HFt-NPs and exposure to the alternating magnetic field. Clear indications of an advanced stage of apoptotic process is also observed from immunocytochemistry analysis. The obtained data suggest this system represents a promising candidate for the development of a protein-based theranostic nanoplatform.

Nanoparticle (NP)-based materials are promising agents for enhancing cancer diagnosis and treatment. Once functionalized for selective targeting of tumor-expressed molecules, they can specifically deliver drugs and diagnostic molecules inside tumor cells. In the present work, we evaluated the in vivo melanoma-targeting ability of a nanovector (HFt-MSH-PEG) based on human protein ferritin (HFt), functionalized with both melanoma-targeting melanoma stimulating hormone (α-MSH) and stabilizing poly(ethylene glycol) (PEG) molecules. Independent and complementary techniques, such as whole-specimen confocal microscopy and magnetic resonance imaging, were used to detect in vivo localization of NP constructs with suitable tracers (i.e., fluorophores or magnetic metals). Targeted HFt-MSH-PEG NPs accumulated persistently at the level of primary melanoma and with high selectivity with respect to other organs. Melanoma localization of untargeted HFt-PEG NPs, which lack the α-MSH moiety, was less pronounced. Furthermore, HFt-MSH-PEG NPs accumulated to a significantly lower extent and with a different distribution in a diverse type of tumor (TS/A adenocarcinoma), which does not express α-MSH receptors. Finally, in a spontaneous lung metastasis model, HFt-MSH-PEG NPs localized at the metastasis level as well. These results suggest that HFt-MSH-PEG NPs are suitable carriers for selective in vivo delivery of diagnostic or therapeutic agents to cutaneous melanoma.

Materials and methods In the present work, we evaluated the in vivo melanomatargeting ability of a nanovector (HFt-MSH-PEG) based on human protein ferritin (HFt), functionalized with both melanoma-targeting melanoma stimulating hormone (a-MSH) and stabilizing poly(ethylene glycol) (PEG) molecules. We used two independent and complementary techniques, such as whole-specimen confocal microscopy and magnetic resonance imaging, to detect the in vivo localization of NP constructs endowed with suitable tracers (i.e., fluorophores or magnetic metals).