Functional characterization of protein PDIA3

PDIA3 protein is a prominent member of the PDI family that has attracted a significant attention by the research community. It is also becoming increasingly evident that PDIA3 could be a pharmacological target in different pathological conditions, though the mechanisms in which it is involved are not completely understood. For this reason, my PhD project puts the spotlight on the PDIA3 involvement in disease processes, focusing on Alzheimer disease and platelet aggregation, and then characterizing its new features and roles in the development of the aforementioned diseases. At present, no selective compounds are known to modulate the PDIA3 biological functions. The discovery of specific PDIA3 interactors could be used in repressing or stimulating PDIA3 in biological model, thus providing useful information on PDIA3 functions

Influence of ellagitannins extracted by pomegranate fruit on disulfide isomerase PDIA3 activity

Pomegranate fruit is a functional food of high interest for human health due to its wide range of phytochemicals with antioxidant properties are implicated in the prevention of inflammation and cancer. Ellagitannins, such as punicalagin and ellagic acid, play a role as anti-atherogenic and neuroprotective molecules in the complex fighting against the degenerative diseases. The aim of this work was to evaluate the composition in punicalagins and ellagic acid of differently obtained extracts from whole fruit, peels and juices, prepared by squeezing or by centrifugation, of pomegranate belonging to different cultivars. Moreover, a wider phenolic fingerprint was also determined. The bioactivity of the extracts was tested on the redox activity of PDIA3 disulfide isomerase, an enzyme involved in the regulation of several cellular functions and associated with different diseases such as cancer, prion disorders, Alzheimer’s and Parkinson’s diseases. The results demonstrate that the different ratios between punicalagin and ellagic acid modulate the enzyme activity and other ellagitannins could interfere with this activity

Shmt2: a stat3 signaling new player in prostate cancer energy metabolism

Prostate cancer (PCa) is a multifactorial disease characterized by the aberrant activity of different regulatory pathways. STAT3 protein mediates some of these pathways and its activation is implicated in the modulation of several metabolic enzymes. A bioinformatic analysis indicated a STAT3 binding site in the upstream region of SHMT2 gene. We demonstrated that in LNCaP, PCa cells' SHMT2 expression is upregulated by the JAK2/STAT3 canonical pathway upon IL-6 stimulation. Activation of SHTM2 leads to a decrease in serine levels, pushing PKM2 towards the nuclear compartment where it can activate STAT3 in a non-canonical fashion that in turn promotes a transient shift toward anaerobic metabolism. These results were also confirmed on FFPE prostate tissue sections at different Gleason scores. STAT3/SHMT2/PKM2 loop in LNCaP cells can modulate a metabolic shift in response to inflammation at early stages of cancer progression, whereas a non-canonical STAT3 activation involving the STAT3/HIF-1α/PKM2 loop is responsible for the maintenance of Warburg effect distinctive of more aggressive PCa cells. Chronic inflammation might thus prime the transition of PCa cells towards more advanced stages, and SHMT2 could represent a missing factor to further understand the molecular mechanisms responsible for the transition of prostate cancer towards a more aggressive phenotyp

ERp57 chaperon protein protects neuronal cells from Aβ-induced toxicity

Alzheimer's disease (AD) is a neurodegenerative disorder whose main pathological hallmark is the accumulation of Amyloid-β peptide (Aβ) in the form of senile plaques. Aβ can cause neurodegeneration and disrupt cognitive functions by several mechanisms, including oxidative stress. ERp57 is a protein disulfide isomerase involved in the cellular stress response and known to be present in the cerebrospinal fluid of normal individuals as a complex with Aβ peptides, suggesting that it may be a carrier protein which prevents aggregation of Aβ. Although several studies show ERp57 involvement in neurodegenerative diseases, no clear mechanism of action has been identified thus far. In this work, we gain insights into the interaction of Aβ with ERp57, with a special focus on the contribution of ERp57 to the defense system of the cell. Here, we show that recombinant ERp57 directly interacts with the Aβ25-35 fragment in vitro with high affinity via two in silico-predicted main sites of interaction. Furthermore, we used human neuroblastoma cells to show that short-term Aβ25-35 treatment induces ERp57 decrease in intracellular protein levels, different intracellular localization, and ERp57 secretion in the cultured medium. Finally, we demonstrate that recombinant ERp57 counteracts the toxic effects of Aβ25-35 and restores cellular viability, by preventing Aβ25-35 aggregation. Overall, the present study shows that extracellular ERp57 can exert a protective effect from Aβ toxicity and highlights it as a possible therapeutic tool in the treatment of AD

PDIA3 expression is altered in the limbic brain regions of triple-transgenic mouse model of Alzheimer's disease

In the present study, we used a mouse model of Alzheimer's disease (AD) (3×Tg-AD mice) to longitudinally analyse the expression level of PDIA3, a protein disulfide isomerase and endoplasmic reticulum (ER) chaperone, in selected brain limbic areas strongly affected by AD-pathology (amygdala, entorhinal cortex, dorsal and ventral hippocampus). Our results suggest that, while in Non-Tg mice PDIA3 levels gradually reduce with aging in all brain regions analyzed, 3×Tg-AD mice showed an age-dependent increase in PDIA3 levels in the amygdala, entorhinal cortex, and ventral hippocampus. A significant reduction of PDIA3 was observed in 3×Tg-AD mice already at 6 months of age, as compared to age-matched Non-Tg mice. A comparative immunohistochemistry analysis performed on 3×Tg-AD mice at 6 (mild AD-like pathology) and 18 (severe AD-like pathology) months of age showed a direct correlation between the cellular level of Aβ and PDIA3 proteins in all the brain regions analysed, even if with different magnitudes. Additionally, an immunohistochemistry analysis showed the presence of PDIA3 in all post-mitotic neurons and astrocytes. Overall, altered PDIA3 levels appear to be age- and/or pathology-dependent, corroborating the ER chaperone's involvement in AD pathology, and supporting the PDIA3 protein as a potential novel therapeutic target for the treatment of AD

Sorcin is an early marker of neurodegeneration, Ca2+ dysregulation and endoplasmic reticulum stress associated to neurodegenerative diseases

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration

Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic

The study of organelle contact sites has received a great impulse due to increased interest in the understanding of their involvement in many disease conditions. Split-GFP-based contact sites (SPLICS) reporters emerged as essential tools to easily detect changes in a wide range of organelle contact sites in cultured cells and in vivo, e.g., in zebrafish larvae. We report here on the generation of a new vector library of SPLICS cloned into a piggyBac system for stable and inducible expression of the reporters in a cell line of interest to overcome any potential weakness due to variable protein expression in transient transfection studies. Stable HeLa cell lines expressing SPLICS between the endoplasmic reticulum (ER) and mitochondria (MT), the ER and plasma membrane (PM), peroxisomes (PO) and ER, and PO and MT, were generated and tested for their ability to express the reporters upon treatment with doxycycline. Moreover, to take advantage of these cellular models, we decided to follow the behavior of different membrane contact sites upon modulating cholesterol traffic. Interestingly, we found that the acute pharmacological inhibition of the intracellular cholesterol transporter 1 (NPC1) differently affects membrane contact sites, highlighting the importance of different interfaces for cholesterol sensing and distribution within the cell

PINK1/Parkin Mediated Mitophagy, Ca2+ Signalling, and ER–Mitochondria Contacts in Parkinson’s Disease

Endoplasmic reticulum (ER)–mitochondria contact sites are critical structures for cellular function. They are implicated in a plethora of cellular processes, including Ca2+ signalling and mitophagy, the selective degradation of damaged mitochondria. Phosphatase and tensin homolog (PTEN)-induced kinase (PINK) and Parkin proteins, whose mutations are associated with familial forms of Parkinson’s disease, are two of the best characterized mitophagy players. They accumulate at ER–mitochondria contact sites and modulate organelles crosstalk. Alterations in ER–mitochondria tethering are a common hallmark of many neurodegenerative diseases including Parkinson’s disease. Here, we summarize the current knowledge on the involvement of PINK1 and Parkin at the ER–mitochondria contact sites and their role in the modulation of Ca2+ signalling and mitophagy

ER-Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches

Organelle intercommunication represents a wide area of interest. Over the last few decades, increasing evidence has highlighted the importance of organelle contact sites in many biological processes including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy but also their involvement in pathological conditions. ER-mitochondria tethering is one of the most investigated inter-organelle communications and it is differently modulated in response to several cellular conditions including, but not limited to, starvation, Endoplasmic Reticulum (ER) stress, and mitochondrial shape modifications. Despite many studies aiming to understand their functions and how they are perturbed under different conditions, approaches to assess organelle proximity are still limited. Indeed, better visualization and characterization of contact sites remain a fascinating challenge. The aim of this review is to summarize strengths and weaknesses of the available methods to detect and quantify contact sites, with a main focus on ER-mitochondria tethering

Punicalagin, an active pomegranate component, is a new inhibitor of pdia3 reductase activity

Background: Polyphenolic compounds isolated from pomegranate fruit possess several pharmacological activities including anti-inflammatory, hepatoprotective, antigenotoxic and anticoagulant activities. The present work focuses the attention on PDIA3 interaction with punicalagin and ellagic acid, the most predominant components of pomegranate extracts. PDIA3, a member of the protein disulfide isomerase family involved in several cellular functions, is associated with different human diseases and it has the potential to be a pharmacological target. Methods: The interaction of polyphenols with PDIA3 purified protein was explored by fluorescence quenching and calorimetric techniques and their effect on PDIA3 activity was investigated. Results: A higher affinity was observed for punicalagin which also strongly affects PDIA3 reductase activity in vitro as a non-competitive inhibitor. Isothermal titration calorimetry confirmed the high affinity of punicalagin for PDIA3. Considering the PDIA3 involvement in oxidative cellular stress response observed in neuroblastoma cells after treatment with hydrogen peroxide, a comparative study was conducted to evaluate the effect of punicalagin on wild type and PDIA3-silenced cells. Punicalagin increases the cell sensitivity to hydrogen peroxide in neuroblastoma cells, but this effect is drastically reduced in PDIA3-silenced cells treated in the same experimental conditions. Conclusions: Punicalagin binds PDIA3 and inhibits its redox activity. Comparative experiments conducted on unsilenced and PDIA3-silenced neuroblastoma cells suggest the potential of punicalagin to modulate PDIA3 reductase activity also in a biological model. General significance: Punicalagin can be used as a new PDIA3 inhibitor and this can provide information on the molecular mechanisms underlying the biological activities of PDIA3 and punicalagin