Investigation of molecular mechanisms involving protein trafficking

The cellular protein traffic and the molecular mechanisms involved in the transport of each individual protein towards a specific destination are crucial events for cell physiology. The 2013 Nobel Prize in Physiology or Medicine honoured three scientists: Dr. Rothman, Dr. Schekman and Dr. Südhof, who have solved the mystery of how the cell organizes its complex transport system. They demonstrated that mechanisms employed by a cell to accomplish vesicle transport and fusion are independent by cell type, and are the same in all eukaryotic organisms. The description of the machinery regulating vesicle traffic, the major transport system in cells, represented a shift in our understanding of how the eukaryotic cell, characterised by a complex internal compartmentalization, organizes the routing of molecules and proteins to various intracellular destinations, as well as to the outside of the cell. The protein traffic plays a very critical role for a variety of physiological processes in which vesicle fusion and molecules delivery must be strictly controlled, such as during hormones and/ or cytokines release. In absence of this wonderful and precise organization, the cell would lapse into chaos. Defective vesicle transport leads to the development of a variety of neurological and immunological disorders, as well as in chronic pathologies, such as the diabetes. Many of these disorders occur in presence of mutation at level of different genes, that often give rise to proteins still catalytically active, but not able to reach the right cell compartment where operate, leading often to the pathological accumulation of metabolite over a toxic threshold. The comprehension at the molecular level how the protein trafficking process occurs in vivo and how it is impaired in pathological conditions might lead to the identification of new targets for therapeutic treatments. In this perspective, my PhD project was focused on the investigation of molecular mechanisms involved in traffic processes of several proteins associated to genetic diseases when they are mutated. These investigations have been carried out basically by using functional proteomic approaches. The role that a specific protein plays in intra- and extra-cellular processes is clarified by the identification of its molecular partners. Indeed, the association of an individual protein, whose its function is unknown, with protein complexes involved in well definite cellular processes would be strongly suggestive of its biological function. A classical functional proteomics approach consists of isolation of protein complexes involving the target protein (bait) from a cell lysate by immunoprecipitation. Proteins so purified were then fractionated by SDS-PAGE, digested in situ with trypsin and identified by nano-LC-MS/MS methodologies integrated with protein database search. In this PhD thesis, the above strategy was employed in the investigation of molecular mechanisms impaired in two rare genetic disorders: Wilson and Pompe disease. Wilson disease, described in Chapter 2, is a disorder characterised by defective copper excretion, due to mutations in copper transporter ATP7B. In normal conditions, ATP7B binds Cu in the trans-Golgi network and moves to the plasma membrane where delivery the Cu in bile channels. Although the most frequent ATP7B mutants, such as ATP7B(H1069Q), potentially are able to bind Cu, they cannot reach the plasma membrane where the excess of Cu has to be removed. In order to evaluate which molecular pathways result altered by expression of ATP7B mutants, a comparison between the interactomes of the wild type protein and the mutant ATP7B(H1069Q) was performed by functional proteomics approach. Pompe disease, discussed in Chapter 3, is a rare disorder of glycogen metabolism caused by mutations in gene encoding GAA, essential enzyme for the degradation of glycogen to glucose in lysosomes. Although the biochemistry GAA activity and genetic basis of the associated disorder are well characterized, the routes followed by wild type GAA to reach lysosomes and impaired in presence of mutants are still unclear. This work was focused on the investigation of the intracellular pathways controlling GAA traffic by the identification of protein partners associated to the enzyme on the route from ER to lysosomes. Similarly, the uptake pathway of recombinant GAA (rhGAA), used in Enzyme Replacement Therapy (ERT), was also studied in order to define the fate of recombinant protein once got into the cell. In Chapter 4, the investigation of molecular mechanisms impaired in amyloidosis has been described. Amyloidosis diseases are associated to the formation of protein aggregates at level of different organs, often associated to the presence of genetic variants of secreted proteins. The study concerning amyloidosis has been carried out by using a double approach: the conformational characterization of some amyloidogenic ApoAI variants was investigated in vitro by a strategy based on complementary proteolysis coupled with mass spectrometry (LC-MS). This methodology allowed clarifying the effect of single point mutations on the protein folding and stability thus to make hypothesis on the structural basis of amyloidosis as protein misfolding/mistrafficking diseases. Moreover a new proteomic strategy for typing amyloid deposits was developed during the period spent abroad at Wolfson Drug Discovery Unit, Centre for Amyloidosis and Acute Phase Proteins, Division of Medicine, University College London (Royal Free Campus), London, UK. Diagnosis and treatment of systemic amyloidosis depends on the correct identification of the amyloid protein triggering the formation of aggregates. In fact, a precise identification of the amyloid fibril protein is essential for the definition of an appropriate therapy. In this field, the proteomic analysis of amyloid deposits provides a chemical characterization of fibrillar constituents, which add important details to genetic sequencing and immunohistochemistry analysis. However, there are ambiguous cases, in which more than one potentially amyloidogenic protein is found within the patient’s biopsy and an accurate diagnosis becomes challenging. The work was aimed at the development of a methodology based on decellularization of human fat biopsies, tryptic digestion and mass spectrometry analysis (LC-MS/MS) in order to eliminate background contamination and improve the specificity of amyloid typing. The use of deoxycholate detergent and the shaking of fat biopsy in tissue lyser resulted fundamental in decellularization procedure. The innovative strategy of decellularization proved to be a simple way to improve the accuracy and specificity of proteomic identification of amyloid fibril type removing most of cellular and plasma proteins background from tissue without altering properties of amyloid fibrils

The Protein Network in Subcutaneous Fat Biopsies from Patients with AL Amyloidosis: More Than Diagnosis?

AL amyloidosis is caused by the misfolding of immunoglobulin light chains leading to an impaired function of tissues and organs in which they accumulate. Due to the paucity of -omics profiles from undissected samples, few studies have addressed amyloid-related damage system wide. To fill this gap, we evaluated proteome changes in the abdominal subcutaneous adipose tissue of patients affected by the AL isotypes κ and λ. Through our retrospective analysis based on graph theory, we have herein deduced new insights representing a step forward from the pioneering proteomic investigations previously published by our group. ECM/cytoskeleton, oxidative stress and proteostasis were confirmed as leading processes. In this scenario, some proteins, including glutathione peroxidase 1 (GPX1), tubulins and the TRiC complex, were classified as biologically and topologically relevant. These and other results overlap with those already reported for other amyloidoses, supporting the hypothesis that amyloidogenic proteins could induce similar mechanisms independently of the main fibril precursor and of the target tissues/organs. Of course, further studies based on larger patient cohorts and different tissues/organs will be essential, which would be a key point that would allow for a more robust selection of the main molecular players and a more accurate correlation with clinical aspects

Amyloid Formation by Globular Proteins: The Need to Narrow the Gap Between in Vitro and in Vivo Mechanisms

The globular to fibrillar transition of proteins represents a key pathogenic event in the development of amyloid diseases. Although systemic amyloidoses share the common characteristic of amyloid deposition in the extracellular matrix, they are clinically heterogeneous as the affected organs may vary. The observation that precursors of amyloid fibrils derived from circulating globular plasma proteins led to huge efforts in trying to elucidate the structural events determining the protein metamorphosis from their globular to fibrillar state. Whereas the process of metamorphosis has inspired poets and writers from Ovid to Kafka, protein metamorphism is a more recent concept. It is an ideal metaphor in biochemistry for studying the protein folding paradigm and investigating determinants of folding dynamics. Although we have learned how to transform both normal and pathogenic globular proteins into fibrillar polymers in vitro, the events occurring in vivo, are far more complex and yet to be explained. A major gap still exists between in vivo and in vitro models of fibrillogenesis as the biological complexity of the disease in living organisms cannot be reproduced at the same extent in the test tube. Reviewing the major scientific attempts to monitor the amyloidogenic metamorphosis of globular proteins in systems of increasing complexity, from cell culture to human tissues, may help to bridge the gap between the experimental models and the actual pathological events in patients

Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation

C. elegans expressing D76N β 2 -microglobulin: a model for in vivo screening of drug candidates targeting amyloidosis

Funder: Ministero dell'Istruzione, dell'Università e della Ricerca Dipartimenti di Eccellenza 2018-2022 grant to the Molecular Medicine Department (University of Pavia)Abstract: The availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates

Identification of p38 MAPK and JNK as New Targets for Correction of Wilson Disease-Causing ATP7B Mutants

Wilson disease (WD) is an autosomal recessive disorder that is caused by the toxic accumulation of copper (Cu) in the liver. The ATP7B gene, which is mutated in WD, encodes a multitransmembrane domain adenosine triphosphatase that traffics from the trans-Golgi network to the canalicular area of hepatocytes, where it facilitates excretion of excess Cu into the bile. Several ATP7B mutations, including H1069Q and R778L that are two of the most frequent variants, result in protein products, which, although still functional, remain in the endoplasmic reticulum. Thus, they fail to reach Cu excretion sites, resulting in the toxic buildup of Cu in the liver of WD patients. Therefore, correcting the location of these mutants by leading them to the appropriate functional sites in the cell should restore Cu excretion and would be beneficial to help large cohorts of WD patients. However, molecular targets for correction of endoplasmic reticulum-retained ATP7B mutants remain elusive. Here, we show that expression of the most frequent ATP7B mutant, H1069Q, activates p38 and c-Jun N-terminal kinase signaling pathways, which favor the rapid degradation of the mutant. Suppression of these pathways with RNA interference or specific chemical inhibitors results in the substantial rescue of ATP7B(H1069Q) (as well as that of several other WD-causing mutants) from the endoplasmic reticulum to the trans-Golgi network compartment, in recovery of its Cu-dependent trafficking, and in reduction of intracellular Cu levels. Conclusion: Our findings indicate p38 and c-Jun N-terminal kinase as intriguing targets for correction of WD-causing mutants and, hence, as potential candidates, which could be evaluated for the development of novel therapeutic strategies to combat WD

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Injectable Antiretroviral Drugs: Back to the Future

Current HIV treatment regimens provide sustained virologic suppression, at least partially restore the immune system and have limited side effects; however, they do not allow viral eradication and they are burdened by daily pill intake with a life-long commitment for the people living with HIV (PHIV). Injectable agents might represent a turning point in the care of PHIV, allowing less frequent administration of antiretroviral treatment (ART), more widespread use of pre-exposure prophylaxis (PrEP) and more stable drug levels in the blood, thus increasing the odds to get closer to end the HIV pandemic. The aim of this manuscript is to give a comprehensive review of injectable antiretrovirals that have been used in the past, which are available now, will be available in the future, and their role in the treatment of HIV infectio

The evolution of an ancient disease: tuberculosis discoveries in the centuries

Tuberculosis (TB) and humans have coexisted for more than 40,000 years.The word "tuberculosis" derives from "tubercle", the histological lesion which appears in the organs, described by Pott in the late Eighteenth century and found, by molecular biology, in human skeletons dating back to 5000 BC.Early description of TB can be found in the writings of ancient India and China and in the Bible.In ancient Greece tuberculosis was not considered contagious, but Aristotle recognized the contagious nature of the pig's and ox's scrofula.The suspicion that phthisis is a contagious disease and that isolation can reduce the risk of transmission was expressed for the first time by the Arabian Avicenna, in his work “The canon of medicine”.In 1699, the Health Council of the Republic of Lucca founded the "sanatorium" concept as place of care and isolation.In 1865 Villemain inoculated tubercular material from a human lymph node into a rabbit, obtaining for the first time the typical tubercular lesions.Some years later, on March 24, 1882, Robert Koch announced to the Berlin Society of Physiology the discovery of Mycobacterium tuberculosis.In the same period Virchow improved awareness of risk factors and correct behaviours among the general population.In 1952 Waksman won the Nobel Prize for the discovery of the first active drug against TB: streptomycin.Nevertheless, drug resistance appeared rapidly some years later and it is still a great challenge in TB fight nowadays