Effect of stress on protein homeostasis mediated by FKBP51 as a possible mechanism underlying stress-related disorders

Homeostasis is a dynamic equilibrium fundamental for a healthy system. A major challenge to homeostasis is environmental stress to which the organism reacts with the stress response. The hypothalamic-pituitary-adrenal (HPA) axis is the main regulator of the stress response that, upon activation, leads to the release of glucocorticoids (GCs). GCs are steroid hormones that exert their function via glucocorticoid receptors (GR). They trigger on one hand the appropriate stress response in the periphery, and, on the other, inhibit the HPA axis itself via negative feedback to restore homeostasis. FK506-binding protein 51 (FKBP51) is a co-chaperone able to modulate the GR, and therefore the HPA axis. Furthermore the expression of FKBP5, the gene coding for FKBP51, is induced by GR activation. In the last decade, increasing evidence has unveiled additional roles of FKBP51 in the regulation of several cellular pathways and functions that are independent from its inhibitory role on GR. Among these, FKBP51 has been shown to link stress signaling to macroautophagy, a lytic type of autophagy pathway. Autophagy represents one of the main mechanisms regulating cellular homeostasis and response to stress. For this reason, in the first part of this doctoral thesis, the role of GR-mediated stress was investigated on two further autophagic pathways: 1) the chaperone-mediated autophagy (CMA), a selective type of lytic autophagy, and 2) the secretory autophagy, an unconventional secretory mechanism regulated by autophagy-related proteins and found to be involved in extracellular signaling of immune response. For this aim, an in vitro approach was adopted using human and murine cell lines that were treated with dexamethasone (Dex), a synthetic GR agonist. For the first pathway, biochemical assays indicated that Dex-induced GR activation enhances CMA-mediated degradation of known CMA target proteins and that this process is dependent on FKBP51. Furthermore, the underlying molecular mechanism could be revealed by co-immunoprecipitation that displayed the co-localization of FKBP51, AKT and PHLPP on lysosomes. With a SILAC-based proteomics analysis, the proteome-wide effect of Dex-induced CMA could be observed and novel CMA targets were identified. For the second pathway, interactome and co-immunoprecipitation analyses revealed the involvement of FKBP51 in the SNARE complex assembly essential for secretory autophagy. Furthermore, treatment with Dex lead to a strengthened interaction between the SNARE proteins and FKBP51, and to an increased secretion of IL1B, a well characterized cargo of secretory autophagy, as observed with in vitro ELISA experiments and in vivo hippocampal microdialyses. A global effect of Dex-induced secretory autophagy was finally observed with a secretome analysis. The second part of my doctoral thesis focused on FKBP5/51 transcription variants and protein isoforms. In fact, despite its involvement in many cellular functions and disorders, very little is known about its four transcription variants and two isoforms. Thus, expression and degradation dynamics of FKBP51 isoforms and their differential functions in known molecular pathways were analyzed. Overall this study highlighted FKBP51 as crucial mediator of the stress response on two autophagic pathways, which might contribute to the regulation of cell and protein homeostasis. Furthermore, this regulatory mechanism might underlie the link of stress to immune and psychiatric disorders

The ‘corporate’ corruption of identity in Tullio Avoledo’s Lo stato dell’ Unione

Since the political upheavals of the early 1990s, Italian writers have continually confronted issues of political identity, particularly in view of the fascistic tendencies that are increasingly polluting society. It is in this context that Tullio Avoledo, a writer from Friuli, continues the strong Italian tradition of impegno, or ‘commitment’. This essay examines his third novel, Lo stato dell’unione (Turin, Einaudi, 2005), addressing specifically the two closely-linked, destabilizing – for the individual – elements of corruption and conspiracy in the business world

Counteracting effects operating on Src homology 2 domain-containing protein-tyrosine phosphatase 2 (SHP2) function drive selection of the recurrent Y62D and Y63C substitutions in Noonan syndrome

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2

Targeting oncogenic Src homology 2 domain-containing phosphatase 2 (SHP2) by inhibiting its protein-protein interactions

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2

Essential of audiology: screening and postscreening

Newborn hearing screening is a type of screening test for the early detection of hearing loss. It can recognize with good accuracy newborns affected by hearing impairment allowing an early diagnosis and intervention and avoiding cognitive and linguistic deficits [1-6]. The incidence of bilateral sensorineural hearing loss (SNHL) in Sicily is 2.35 cases per 1000 newborns; this value increases to 2.95 if we consider also unilateral SNHL [2,3] and to 10 cases per 1000 births among infants at risk [7-9]. A correct newborn hearing screening programme is based on different protocols depending on the presence/ absence of audiologic risk factor

Impact of patent ductus arteriosus on non-invasive assessments of lung fluids in very preterm infants during the transitional period

This prospective observational study aimed to evaluate whether lung fluids, assessed by lung ultrasonography and transthoracic electrical bioimpedance (TEB), may be influenced by the presence of a haemodynamically significant patent ductus arteriosus (hsPDA) in very preterm infants during the transitional period. Infants < 32 weeks of gestational age (GA) admitted to the neonatal intensive care units of IRCCS AOU Bologna and Niguarda Metropolitan Hospital of Milan (Italy) underwent a daily assessment of a lung ultrasound score (LUS) and of a TEB-derived index of thoracic fluid contents (TFC) during the first 72 h after birth. Echocardiographic scans were simultaneously performed to evaluate the concomitant ductal status (hsPDA vs. restrictive or closed duct). The correlation between LUS, TFC, and the ductal status was tested using generalized estimating equations. Forty-six infants (median GA: 29 [interquartile range, IQR: 27-31] weeks; median birth weight: 1099 [IQR: 880-1406] g) were included. At each daily evaluation, the presence of a hsPDA was associated with significantly higher LUS and TFC compared with a restrictive or closed ductus (p < 0.01 for all comparisons). These results were confirmed significant even after adjustment for GA and for the ongoing modality of respiratory support.Conclusion: Even during the first 72 h of life, the presence of a hsPDA determines a significant increase in pulmonary fluids which can be non-invasively detected and monitored over time using lung ultrasonography and TEB

Variable susceptibility of laboratory strains of Aspergillus nidulans to hygromycin B and other ribosomal antibiotics

Variable susceptibility of laboratory strains of Aspergillus nidulans to hygromycin and other ribosomal antibiotics