INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS

Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.

STRUCTURAL AND FUNCTIONAL STUDIES OF CYCLIC K48-LINKED DIUBIQUITIN

K48-linked di-ubiquitin exists in a dynamic equilibrium between open and closed states. The structure of K48-Ub2 in the closed conformation features a hydrophobic interface formed between the two Ub domains. The same hydrophobic residues at the interface are involved in binding to ubiquitin-associated (UBA) domains. Cyclization of K48-Ub2 should limit the range of conformations available for such interactions. Interestingly, cyclic K48-linked Ub2 (cycUb2) has been found in vivo and can be isolated in vitro to study its structure and dynamics. In this study, a crystal structure of cycUb2 was obtained, and the dynamics of cycUb2 were characterized by solution NMR. The crystal structure of cycUb2, which is in agreement with solution NMR data, is closed with the hydrophobic patches of each Ub domain buried at the interface. Despite its structural constraints, cycUb2 was still able to interact with UBA domains, albeit with lower affinity.

Epothilone-d rescues cognition and attenuates alzheimer’s disease-like pathology in APP/PS1 mice

AIMS: Cognitive decline in Alzheimer's disease (AD) patients has been linked to synaptic damage and neuronal loss. Hyperphosphorylation of tau protein destabilizes microtubules leading to the accumulation of autophagy/vesicular material and the generation of dystrophic neurites, thus contributing to axonal/synaptic dysfunction. In this study, we analyzed the effect of a microtubule-stabilizing compound in the progression of the disease in the hippocampus of APP751SL/PS1M146L transgenic model. METHODS: APP/PS1 mice (3 month-old) were treated with a weekly intraperitoneal injection of 2 mg/kg epothilone-D (Epo-D) for 3 months. Vehicle-injected animals were used as controls. Mice were tested on the Morris water maze, Y-maze and object-recognition tasks for memory performance. Abeta, AT8, ubiquitin and synaptic markers levels were analyzed by Western-blots. Hippocampal plaque, synaptic and dystrophic loadings were quantified by image analysis after immunohistochemical stainings. RESULTS: Epo-D treated mice exhibited a significant improvement in the memory tests compared to controls. The rescue of cognitive deficits was associated to a significant reduction in the AD-like hippocampal pathology. Levels of Abeta, APP and ubiquitin were significantly reduced in treated animals. This was paralleled by a decrease in the amyloid burden, and more importantly, in the plaque-associated axonal dystrophy pathology. Finally, synaptic levels were significantly restored in treated animals compared to controls. CONCLUSION: Epo-D treatment promotes synaptic and spatial memory recovery, reduces the accumulation of extracellular Abeta and the associated neuritic pathology in the hippocampus of APP/PS1 model. Therefore, microtubule stabilizing drugs could be considered therapeutical candidates to slow down AD progression. Supported by FIS-PI12/01431 and PI15/00796 (AG),FIS-PI12/01439 and PI15/00957(JV)

VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin

Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.08 to -6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the P(RB) promoter, as indicated by analysis of peptidoglycan precursors and of VanX(B) D,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the P(YB) resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the P(RB) regulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanS(B) gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the P(RB) promoter in the absence of vancomycin.

Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein

Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer's disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.

Whole Exome Sequencing widens the genetic landscape of Hereditary Optic Neuropathies

Hereditary optic neuropathies (HON) are a genetic cause of visual impairment characterized by degeneration of retinal ganglion cells. The majority of HON are caused by pathogenic variants in mtDNA genes and in gene OPA1. However, several other genes can cause optic atrophy and can only be identified by high throughput genetic analysis. Whole Exome Sequencing (WES) is becoming the primary choice in rare disease molecular diagnosis, being both cost effective and informative. We performed WES on a cohort of 106 cases, of which 74 isolated ON patients (ON) and 32 syndromic ON patients (sON). The total diagnostic yield amounts to 27%, slightly higher for syndromic ON (31%) than for isolated ON (26%). The majority of genes found are related to mitochondrial function and already reported for harbouring HON pathogenic variants: ACO2, AFG3L2, C19orf12, DNAJC30, FDXR, MECR, MTFMT, NDUFAF2, NDUFB11, NDUFV2, OPA1, PDSS1, SDHA, SSBP1, and WFS1. Among these OPA1, ACO2, and WFS1 were confirmed as the most relevant genetic causes of ON. Moreover, several genes were identified, especially in sON patients, with direct impairment of non-mitochondrial molecular pathways: from autophagy and ubiquitin system (LYST, SNF8, WDR45, UCHL1), to neural cells development and function (KIF1A, GFAP, EPHB2, CACNA1A, CACNA1F), but also vitamin metabolism (SLC52A2, BTD), cilia structure (USH2A), and nuclear pore shuttling (NUTF2). Functional validation on yeast model was performed for pathogenic variants detected in MECR, MTFMT, SDHA, and UCHL1 genes. For SDHA and UCHL1 also muscle biopsy and fibroblast cell lines from patients were analysed, pointing to possible pathogenic mechanisms that will be investigated in further studies. In conclusion, WES proved to be an efficient tool when applied to our ON cohort, for both common disease-genes identification and novel genes discovery. It is therefore recommended to consider WES in ON molecular diagnostic pipeline, as for other rare genetic diseases.

Hetero-bifunctional molecules as possible therapeutics for the treatment of Alzheimer’s disease.

Alzheimer’s disease (AD) is the most common form of dementia, currently affecting more than 50 million people worldwide. In recent years attention towards this disease has risen in search for discovery and development of a drug that can stop it. Indeed, therapies for AD provide only temporary symptomatic relief. The cause for the high attrition rate for AD drug discovery has been attributed to several factors, including the fact that the AD pathogenesis is not yet fully understood. Nevertheless, what is increasingly recognized is that AD is a multifactorial syndrome, characterized by many conditions which may lead to neuronal death. Given this, it is widely accepted that a molecule able to modulate more than one target would bring benefit to the therapy of AD. In the first chapter of this thesis, there are reported two projects regarding the design and synthesis of new series of GSK-3/HDAC dual inhibitors, two of the main enzymes involved in AD. Two different series of compounds were synthesized and evaluated for their inhibitory activity towards the target enzymes. The best compounds of the series were selected for further biologic investigation to evaluate their properties. The second project focused on the design of non ATP-competitive GSK-3 inhibitors combined with HDAC inhibition properties. Also in this case, the best compounds of the series were selected for biologic investigation to further evaluate their properties. In chapter 2, the design and synthesis of a GSK-3-directed Proteolysis Targeting Chimeras (PROTAC), a new technology in drug discovery that act through degradation rather than inhibition, is reported. The design and synthesis of a small series of GSK-3-directed PROTACs was achieved. In vitro assays were performed to evaluate the GSK-3-degradation ability, the effective involvement of E3 ubiquitine ligase in the process and their neuroprotective abilities.