SciCombinator

Discover the most talked about and latest scientific content & concepts.

Concept: Endoplasmic reticulum

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O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism.The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.

Concepts: Plasmodium vivax, Anopheles, Plasmodium, Plasmodium falciparum, Immune system, Endoplasmic reticulum, Apicomplexa, Malaria

71

Diabetes and its concurrent complications impact a significant proportion of the population of the US and create a large financial burden on the American health care system. FDA-approved maggot debridement therapy (MDT), the application of sterile laboratory-reared Lucilia sericata (green bottle fly) larvae to wounds, is a cost-effective and successful treatment for diabetic foot ulcers and other medical conditions. Human platelet derived growth factor-BB (PDGF-BB) is a secreted dimeric peptide growth factor that binds the PDGF receptor. PDGF-BB stimulates cell proliferation and survival, promotes wound healing, and has been investigated as a possible topical treatment for non-healing wounds. Genetic engineering has allowed for expression and secretion of human growth factors and other proteins in transgenic insects. Here, we present a novel concept in MDT technology that combines the established benefits of MDT with the power of genetic engineering to promote healing. The focus of this study is to create and characterize strains of transgenic L. sericata that express and secrete PDGF-BB at detectable levels in adult hemolymph, whole larval lysate, and maggot excretions/ secretions (ES), with potential for clinical utility in wound healing.

Concepts: Endoplasmic reticulum, Calliphoridae, Secretion, Green bottle fly, Signal transduction, Wound healing, Platelet-derived growth factor, Cell

67

Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction.

Concepts: Mitochondrion, Organelle, Metabolism, Proteins, Signal transduction, Cell, Endoplasmic reticulum

63

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade’s refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Concepts: Leptospira, Endoplasmic reticulum, Amino acid, Microbiology, DNA, Evolution, Gene, Bacteria

61

Persistent exposure to man-made endocrine disrupting chemicals during fetal endocrine development may lead to disruption of metabolic homeostasis contributing to childhood obesity. Limited cellular platforms exist to test endocrine disrupting chemical-induced developmental abnormalities in human endocrine tissues. Here we use an human-induced pluripotent stem cell-based platform to demonstrate adverse impacts of obesogenic endocrine disrupting chemicals in the developing endocrine system. We delineate the effects upon physiological low-dose exposure to ubiquitous endocrine disrupting chemicals including, perfluoro-octanoic acid, tributyltin, and butylhydroxytoluene, in endocrine-active human-induced pluripotent stem cell-derived foregut epithelial cells and hypothalamic neurons. Endocrine disrupting chemicals induce endoplasmic reticulum stress, perturb NF-κB, and p53 signaling, and diminish mitochondrial respiratory gene expression, spare respiratory capacity, and ATP levels. As a result, normal production and secretion of appetite control hormones, PYY, α-MSH, and CART, are hampered. Blocking NF-κB rescues endocrine disrupting chemical-induced aberrant mitochondrial phenotypes and endocrine dysregulation, but not ER-stress and p53-phosphorylation changes.Harmful chemicals that disrupt the endocrine system and hormone regulation have been associated with obesity. Here the authors apply a human pluripotent stem cell-based platform to study the effects of such compounds on developing gut endocrine and neuroendocrine systems.

Concepts: Metabolism, Endoplasmic reticulum, Endocrinology, Endocrine system, Developmental biology, Obesity, Endocrine disruptor, Cell

55

The recent Zika virus (ZIKV) outbreak in the Western hemisphere is associated with severe pathology in newborns, including microcephaly and brain damage. The mechanisms underlying these outcomes are under intense investigation. Here, we show that a 2015 ZIKV isolate replicates in multiple cell types, including primary human fetal neural progenitors (hNPs). In immortalized cells, ZIKV is cytopathic and grossly rearranges endoplasmic reticulum membranes similar to other flaviviruses. In hNPs, ZIKV infection has a partial cytopathic phase characterized by cell rounding, pyknosis, and activation of caspase 3. Despite notable cell death, ZIKV did not activate a cytokine response in hNPs. This lack of cell intrinsic immunity to ZIKV is consistent with our observation that virus replication persists in hNPs for at least 28 days. These findings, supported by published fetal neuropathology, establish a proof-of-concept that neural progenitors in the developing human fetus can be direct targets of detrimental ZIKV-induced pathology.

Concepts: Infant, Cell, Fetus, Pregnancy, Embryo, Apoptosis, Endoplasmic reticulum

53

During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA-like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated.

Concepts: Infectious disease, Gene, Transmissible spongiform encephalopathy, Endoplasmic reticulum, Protein folding, Proteins, Prion, Protein

49

The bacterial Type VI secretion system (T6SS) delivers proteins into target cells using fast contraction of a long sheath anchored to the cell envelope and wrapped around an inner Hcp tube associated with the secreted proteins. Mechanisms of sheath assembly and length regulation are unclear. Here we study these processes using spheroplasts formed from ampicillin-treated Vibrio cholerae. We show that spheroplasts secrete Hcp and deliver T6SS substrates into neighbouring cells. Imaging of sheath dynamics shows that the sheath length correlates with the diameter of spheroplasts and may reach up to several micrometres. Analysis of sheath assembly after partial photobleaching shows that subunits are exclusively added to the sheath at the end that is distal from the baseplate and cell envelope attachment. We suggest that this mode of assembly is likely common for all phage-like contractile nanomachines, because of the conservation of the structures and connectivity of sheath subunits.

Concepts: Bacteria, Cell biology, Cytosol, Golgi apparatus, Cell, Endoplasmic reticulum, Microbiology, Secretion

43

We surveyed the “dark” proteome-that is, regions of proteins never observed by experimental structure determination and inaccessible to homology modeling. For 546,000 Swiss-Prot proteins, we found that 44-54% of the proteome in eukaryotes and viruses was dark, compared with only ∼14% in archaea and bacteria. Surprisingly, most of the dark proteome could not be accounted for by conventional explanations, such as intrinsic disorder or transmembrane regions. Nearly half of the dark proteome comprised dark proteins, in which the entire sequence lacked similarity to any known structure. Dark proteins fulfill a wide variety of functions, but a subset showed distinct and largely unexpected features, such as association with secretion, specific tissues, the endoplasmic reticulum, disulfide bonding, and proteolytic cleavage. Dark proteins also had short sequence length, low evolutionary reuse, and few known interactions with other proteins. These results suggest new research directions in structural and computational biology.

Concepts: Protein structure, Bacteria, Evolution, DNA, Cell, Bioinformatics, Endoplasmic reticulum, Protein

43

Critical aspects of HIV-1 infection occur in mucosal tissues, particularly in the gut, which contains large numbers of HIV-1 target cells that are depleted early in infection. We used electron tomography (ET) to image HIV-1 in gut-associated lymphoid tissue (GALT) of HIV-1-infected humanized mice, the first three-dimensional ultrastructural examination of HIV-1 infection in vivo. Human immune cells were successfully engrafted in the mice, and following infection with HIV-1, human T cells were reduced in GALT. Virions were found by ET at all stages of egress, including budding immature virions and free mature and immature viruses. Immuno-electron microscopy verified the virions were HIV-1 and showed CD4 sequestration in the endoplasmic reticulum of infected cells. Observation of HIV-1 in infected GALT tissue revealed that most HIV-1-infected cells, identified by immunolabeling and/or the presence of budding virions, were localized to intestinal crypts with pools of free virions concentrated in spaces between cells. Fewer infected cells were found in mucosal regions and the lamina propria. The preservation quality of reconstructed tissue volumes allowed details of budding virions, including structures interpreted as host-encoded scission machinery, to be resolved. Although HIV-1 virions released from infected cultured cells have been described as exclusively mature, we found pools of both immature and mature free virions within infected tissue. The pools could be classified as containing either mostly mature or mostly immature particles, and analyses of their proximities to the cell of origin supported a model of semi-synchronous waves of virion release. In addition to HIV-1 transmission by pools of free virus, we found evidence of transmission via virological synapses. Three-dimensional EM imaging of an active infection within tissue revealed important differences between cultured cell and tissue infection models and furthered the ultrastructural understanding of HIV-1 transmission within lymphoid tissue.

Concepts: Infection, Bacteria, White blood cell, Protein, Virus, Immune system, Lymphatic system, Endoplasmic reticulum