Integrated transcriptomics uncovers an enhanced association between the prion protein gene expression and vesicle dynamics signatures in glioblastomas.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor that exhibits resistance to current treatment, making the identification of novel therapeutic targets essential. In this context, cellular prion protein (PrPC) stands out as a potential candidate for new therapies. Encoded by the PRNP gene, PrPC can present increased expression levels in GBM, impacting cell proliferation, growth, migration, invasion and stemness. Nevertheless, the exact molecular mechanisms through which PRNP/PrPC modulates key aspects of GBM biology remain elusive. METHODS: To elucidate the implications of PRNP/PrPC in the biology of this cancer, we analyzed publicly available RNA sequencing (RNA-seq) data of patient-derived GBMs from four independent studies. First, we ranked samples profiled by bulk RNA-seq as PRNPhigh and PRNPlow and compared their transcriptomic landscape. Then, we analyzed PRNP+ and PRNP- GBM cells profiled by single-cell RNA-seq to further understand the molecular context within which PRNP/PrPC might function in this tumor. We explored an additional proteomics dataset, applying similar comparative approaches, to corroborate our findings. RESULTS: Functional profiling revealed that vesicular dynamics signatures are strongly correlated with PRNP/PrPC levels in GBM. We found a panel of 73 genes, enriched in vesicle-related pathways, whose expression levels are increased in PRNPhigh/PRNP+ cells across all RNA-seq datasets. Vesicle-associated genes, ANXA1, RAB31, DSTN and SYPL1, were found to be upregulated in vitro in an in-house collection of patient-derived GBM. Moreover, proteome analysis of patient-derived samples reinforces the findings of enhanced vesicle biogenesis, processing and trafficking in PRNPhigh/PRNP+ GBM cells. CONCLUSIONS: Together, our findings shed light on a novel role for PrPC as a potential modulator of vesicle biology in GBM, which is pivotal for intercellular communication and cancer maintenance. We also introduce GBMdiscovery, a novel user-friendly tool that allows the investigation of specific genes in GBM biology.

Targeting trafficking as a therapeutic avenue for misfolded GPCRs leading to endocrine diseases.

G protein-coupled receptors (GPCRs) are plasma membrane proteins associated with an array of functions. Mutations in these receptors lead to a number of genetic diseases, including diseases involving the endocrine system. A particular subset of loss-of-function mutant GPCRs are misfolded receptors unable to traffic to their site of function (i.e. the cell surface plasma membrane). Endocrine disorders in humans caused by GPCR misfolding include, among others, hypo- and hyper-gonadotropic hypogonadism, morbid obesity, familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, X-linked nephrogenic diabetes insipidus, congenital hypothyroidism, and familial glucocorticoid resistance. Several in vitro and in vivo experimental approaches have been employed to restore function of some misfolded GPCRs linked to endocrine disfunction. The most promising approach is by employing pharmacological chaperones or pharmacoperones, which assist abnormally and incompletely folded proteins to refold correctly and adopt a more stable configuration to pass the scrutiny of the cell's quality control system, thereby correcting misrouting. This review covers the most important aspects that regulate folding and traffic of newly synthesized proteins, as well as the experimental approaches targeted to overcome protein misfolding, with special focus on GPCRs involved in endocrine diseases.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.

From cartoons to quantitative models in Golgi transport.

BACKGROUND: Cell biology is evolving to become a more formal and quantitative science. In particular, several mathematical models have been proposed to address Golgi self-organisation and protein and lipid transport. However, most scientific articles about the Golgi apparatus are still using static cartoons that miss the dynamism of this organelle. RESULTS: In this report, we show that schematic drawings of Golgi trafficking can be easily translated into an agent-based model using the Repast platform. The simulations generate an active interplay among cisternae and vesicles rendering quantitative predictions about Golgi stability and transport of soluble and membrane-associated cargoes. The models can incorporate complex networks of molecular interactions and chemical reactions by association with COPASI, a software that handles ordinary differential equations. CONCLUSIONS: The strategy described provides a simple, flexible and multiscale support to analyse Golgi transport. The simulations can be used to address issues directly linked to the mechanism of transport or as a way to incorporate the complexity of trafficking to other cellular processes that occur in dynamic organelles. SIGNIFICANCE: We show that the rules implicitly present in most schematic representations of intracellular trafficking can be used to build dynamic models with quantitative outputs that can be compared with experimental results.

Overcoming Biological Barriers With Block Copolymers-Based Self-Assembled Nanocarriers. Recent Advances in Delivery of Anticancer Therapeutics.

Cancer is one of the most common life-threatening illness and it is the world's second largest cause of death. Chemotherapeutic anticancer drugs have many disadvantages, which led to the need to develop novel strategies to overcome these shortcomings. Moreover, tumors are heterogenous in nature and there are various biological barriers that assist in treatment reisistance. In this sense, nanotechnology has provided new strategies for delivery of anticancer therapeutics. Recently, delivery platforms for overcoming biological barriers raised by tumor cells and tumor-bearing hosts have been reported. Among them, amphiphilic block copolymers (ABC)-based self-assembled nanocarriers have attracted researchers worldwide owing to their unique properties. In this work, we addressed different biological barriers for effective cancer treatment along with several strategies to overcome them by using ABC-based self-assembled nanostructures, with special emphasis in those that have the ability to act as responsive nanocarriers to internal or external environmental clues to trigger release of the payload. These nanocarriers have shown promising properties to revolutionize cancer treatment and diagnosis, but there are still challenges for their successful translation to clinical applications.

The Role of the Flagellar Protein FlgJ in the Virulence of Brucella abortus.

Brucella abortus is a facultative intracellular pathogen that causes a zoonosis called brucellosis. This disease leads to abortion and infertility in cattle, and diverse complications in humans. B. abortus is a successful intracellular bacterium that has developed the ability to evade the host's immune system and it replicates in professional and non-professional phagocytic cells, persisting in the different tissues, and organs of its hosts. It has been described that Brucella expresses a polar flagellum under certain conditions, but its function is still unknown. In this study we evaluated the role of the FlgJ, a protein, presumably a peptidoglycan hydrolase involved in flagellum formation and in the virulence of B. abortus strain 2308. B. abortus 2308 ΔflgJ mutant and complemented strains were constructed to study the function of the FlgJ protein in the context of the virulence of this pathogen in in vitro and in vivo assays. The results showed that the elimination of the flgJ gene delays the growth rate of B. abortus in culture, reduces its intracellular survival capacity in professional and non-professional phagocytic cells, rendering it unable to escape from the endocytic route and not reaching the endoplasmic reticulum. It also negatively affects their persistence in BALB/c mice. Functionally, the B. abortus 2308 flgJ gene restored motility to an E. coli flgJ mutant gene. Furthermore, it was discovered that the production of FlgJ protein is associated with the bacterial adherence by B. abortus. Therefore, although the specific function of the polar flagellum for Brucella is unknown, the data indicates that the flagellar flgJ gene and its product are required for full virulence of B. abortus 2308, since its deletion significantly reduces the fitness of this pathogen in vitro and in vivo.

Rab39a and Rab39b Display Different Intracellular Distribution and Function in Sphingolipids and Phospholipids Transport.

Rab GTPases define the identity and destiny of vesicles. Some of these small GTPases present isoforms that are expressed differentially along developmental stages or in a tissue-specific manner, hence comparative analysis is difficult to achieve. Here, we describe the intracellular distribution and function in lipid transport of the poorly characterized Rab39 isoforms using typical cell biology experimental tools and new ones developed in our laboratory. We show that, despite their amino acid sequence similarity, Rab39a and Rab39b display non-overlapping intracellular distribution. Rab39a localizes in the late endocytic pathway, mainly at multivesicular bodies. In contrast, Rab39b distributes in the secretory network, at the endoplasmic reticulum/cis-Golgi interface. Therefore, Rab39a controls trafficking of lipids (sphingomyelin and phospholipids) segregated at multivesicular bodies, whereas Rab39b transports sphingolipids biosynthesized at the endoplasmic reticulum-Golgi factory. Interestingly, lyso bis-phosphatidic acid is exclusively transported by Rab39a, indicating that both isoforms do not exert identical functions in lipid transport. Conveniently, the requirement of eukaryotic lipids by the intracellular pathogen Chlamydia trachomatis rendered useful for dissecting and distinguishing Rab39a- and Rab39b-controlled trafficking pathways. Our findings provide comparative insights about the different subcellular distribution and function in lipid transport of the two Rab39 isoforms.

A mathematical model relates intracellular TLR4 oscillations to sepsis progression.

OBJECTIVE: Oscillations of physiological parameters describe many biological processes and their modulation is determinant for various pathologies. In sepsis, toll-like receptor 4 (TLR4) is a key sensor for signaling the presence of Gram-negative bacteria. Its intracellular trafficking rates shift the equilibrium between the pro- and anti-inflammatory downstream signaling cascades, leading to either the physiological resolution of the bacterial stimulation or to sepsis. This study aimed to evaluate the effects of TLR4 increased expression and intracellular trafficking on the course and outcome of sepsis. RESULTS: Using a set of three differential equations, we defined the TLR4 fluxes between relevant cell organelles. We obtained three different regions in the phase space: (1) a limit-cycle describing unstimulated physiological oscillations, (2) a fixed-point attractor resulting from moderate LPS stimulation that is resolved and (3) a double-attractor resulting from sustained LPS stimulation that leads to sepsis. We used this model to describe available hospital data of sepsis patients and we correctly characterize the clinical outcome of these patients.

Intracellular trafficking of a dynein-based nanoparticle designed for gene delivery.

The success of viruses in the delivery of the viral genome to target cells relies on the evolutionary selection of protein-based domains able to hijack the intermolecular interactions through which cells respond to intra- and extracellular stimuli. In an effort to mimic viral infection capabilities during non-viral gene delivery, a modular recombinant protein named T-Rp3 was recently developed, containing a DNA binding domain, a dynein molecular motor interacting domain, and a TAT-derived transduction domain. Here, we analyzed at the microscopic level the mechanisms behind the cell internalization and intracellular trafficking of this highly efficient modular protein vector. We found that the protein has the ability to self-assemble in discrete protein nanoparticles resembling viral capsids, to bind and condense plasmid DNA (pDNA), and to interact with eukaryotic cell membranes. Confocal and single particle tracking assays performed on living HeLa cells revealed that the T-Rp3 nanoparticles promoted an impressive speed of cellular uptake and perinuclear accumulation. Finally, the protein demonstrated to be a versatile vector, delivering siRNA at efficiencies comparable to Lipofectamine™. These results demonstrate the high potential of recombinant modular proteins with merging biological functions to fulfill several requirements needed to obtain cost-effective non-viral vectors for gene-based therapies.

Fate of Bacillus cereus within phagocytic cells.

In this study we assessed the interaction of different strains of Bacillus cereus with murine peritoneal macrophages and cultured phagocytic cells (Raw 264.7 cells). Association, internalization, intracellular survival, routing of bacteria to different compartments and expression of MHCII were assessed in cells infected with different strains of B. cereus in vegetative form. Association values (adhering + internalized bacteria) and phagocytosis were higher for strain B10502 than those for strains 2 and M2. However, after 90 min interaction, intracellular survival was higher for strain 2 than for strains M2 and B10502. Acquisition of lysosomal markers by B. cereus containing vacuoles (BcCV), assessed by LAMP1 and Lysotracker labelling occurred shortly after internalization. The highest ratio of LAMP1(+)-BcCV was found for strain M2. This strain was able to survive longer than strain B10502 which routes to LAMP1 containing vacuoles to a lesser extent. In addition, strain M2 stimulated expression of MHCII by infected cells. Confocal analyses 60 or 90 min post-infection showed different percentages of co-localization of bacteria with Lysotracker. Results suggest strain-dependent interaction and intracellular killing of B. cereus by phagocytic cells. These findings could be relevant for the pathogenic potential of Bacillus cereus strains.

CFTR Modulators: Shedding Light on Precision Medicine for Cystic Fibrosis.

Cystic fibrosis (CF) is the most common life-threatening monogenic disease afflicting Caucasian people. It affects the respiratory, gastrointestinal, glandular and reproductive systems. The major cause of morbidity and mortality in CF is the respiratory disorder caused by a vicious cycle of obstruction of the airways, inflammation and infection that leads to epithelial damage, tissue remodeling and end-stage lung disease. Over the past decades, life expectancy of CF patients has increased due to early diagnosis and improved treatments; however, these patients still present limited quality of life. Many attempts have been made to rescue CF transmembrane conductance regulator (CFTR) expression, function and stability, thereby overcoming the molecular basis of CF. Gene and protein variances caused by CFTR mutants lead to different CF phenotypes, which then require different treatments to quell the patients' debilitating symptoms. In order to seek better approaches to treat CF patients and maximize therapeutic effects, CFTR mutants have been stratified into six groups (although several of these mutations present pleiotropic defects). The research with CFTR modulators (read-through agents, correctors, potentiators, stabilizers and amplifiers) has achieved remarkable progress, and these drugs are translating into pharmaceuticals and personalized treatments for CF patients. This review summarizes the main molecular and clinical features of CF, emphasizes the latest clinical trials using CFTR modulators, sheds light on the molecular mechanisms underlying these new and emerging treatments, and discusses the major breakthroughs and challenges to treating all CF patients.

HIV-1 Nef: Taking Control of Protein Trafficking.

The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host-cell-interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV-1 virions.

VirJ Is a Brucella Virulence Factor Involved in the Secretion of Type IV Secreted Substrates.

The VirB secretion apparatus in Brucella belongs to the type IV secretion systems present in many pathogenic bacteria and is absolutely necessary for the efficient evasion of the Brucella-containing vacuole from the phagocytic route in professional phagocytes. This system is responsible for the secretion of a plethora of effector proteins that alter the biology of the host cell and promote the intracellular replication process. Although many VirB substrates have been identified in Brucella, we still know very little about the secretion mechanism that mediates their translocation across the two membranes and the periplasmic space. In this manuscript, we describe the identification of a gene, virJ, that codes for a protein with periplasmic localization that is involved in the intracellular replication process and virulence in mice. Our analysis revealed that this protein is necessary for the secretion of at least two VirB substrates that have a periplasmic intermediate and that it directly interacts with them. We additionally show that VirJ also associates with the apparatus per se and that its absence affects the assembly of the complex. We hypothesize that VirJ is part of a secretion platform composed of the translocon and several secretion substrates and that it probably coordinates the proper assembly of this macromolecular complex.

Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor.

Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef.

Structural insights into functional overlapping and differentiation among myosin V motors.

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contribute to understanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors.