scholarly journals The Road Less Traveled? Unconventional Protein Secretion at Parasite–Host Interfaces

2021 ◽  
Vol 9 ◽  
Author(s):  
Erina A. Balmer ◽  
Carmen Faso
Keyword(s):  
Protein Secretion ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Cell Model ◽  
Signal Sequence ◽  
Model Organism ◽  
Secreted Proteins ◽  
Current Status ◽  
Secretory Proteins ◽  
Secretion Pathways

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.

scholarly journals Quality control of glycosylphosphatidylinositol anchor attachment in mammalian cells: a biochemical study

1997 ◽  
Vol 321 (3) ◽  
pp. 655-664 ◽  
Author(s):  
Linda J. WAINWRIGHT ◽  
Mark C. FIELD
Keyword(s):  
Quality Control ◽  
Mammalian Cells ◽  
Biochemical Study ◽  
Signal Sequence ◽  
Sharp Decrease ◽  
Disulphide Bond ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Gpi Anchor

hGHDAF28 is a chimaeric protein consisting of human growth hormone fused to a crippled signal sequence for glycosylphosphatidylinositol (GPI)-anchor addition from decay-accelerating factor, and serves as a model for quality control of GPI-anchor addition. hGHDAF28 is retained in a pre-Golgi compartment and degraded intracellularly by a mechanism with similarity to that for other endoplasmic reticulum (ER)-retained proteins (Field, Moran, Lee, Keller and Caras (1994) J. Biol. Chem. 269, 10830Ő10837). We have studied the specific pathway of degradation for hGHDAF28 using a number of compounds which affect protein folding and trafficking pathways in eukaryotic cells. We found that high concentrations of dithiothreitol (DTT) accelerated loss of hGHDAF28 by degradation from cell lysates, without promoting secretion or alteration of disulphide-bond distribution, in contrast to a number of other examples of ER-retained proteins where DTT alters disulphide-bond formation. Additionally, degradation of hGHDAF28 was sensitive to pH, being promoted at pH 6.0 and inhibited at pH 8.0; however, the latter effect was transient, indicating incomplete blockade. Degradation was also partially enhanced by depletion of ER calcium with thapsigargin, but this was again a partial and transient effect. Furthermore, degradation was temperature sensitive, with a gradual decrease in rate observed at lower temperatures. However, a sharp decrease in turnover between 15 ŶC and 20 ŶC, indicative of a requirement for transport to a post-ER compartment, was not observed. Degradation of hGHDAF28 was insensitive to treatment with nocodozole or compounds preventing cytoplasmic autophagy, suggesting that ER degradation is independent of classical autophagy and microtubule-dependent processes. In addition, disruption of N-glycosylation with tunicamycin, or inhibition of processing of immature N-glycan chains with castanospermine or deoxynojirimycin, had little effect on the stability of hGHDAF28, suggesting that disruption of the BiP/calnexin quality-control system by bulk cellular secretory proteins does not influence the ER-degradation pathway of hGHDAF28. Intermolecular hGHDAF28 cysteine bonds result in the formation of aggregates which are probably important in the retention of the molecule. The insensitivity of this structure to reduction in vivo, together with the enhanced degradation rate, indicates that DTT mediates its effect on stability via a molecule involved in degradation of hGHDAF28, possibly a thiol-sensitive protease.

scholarly journals Infection-Blocking Genes of a Symbiotic Rhizobium leguminosarum Strain That Are Involved in Temperature-Dependent Protein Secretion

2003 ◽  
Vol 16 (1) ◽  
pp. 53-64 ◽  
Author(s):  
M. R. Bladergroen ◽  
K. Badelt ◽  
H. P. Spaink
Keyword(s):  
Nitrogen Fixation ◽  
Protein Secretion ◽  
Rhizobium Leguminosarum ◽  
Signal Sequence ◽  
Gene Clusters ◽  
Secreted Proteins ◽  
Type I ◽  
Secretion Systems ◽  
Temperature Dependent ◽  
Animal Pathogens

Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.

scholarly journals Advances and perspectives in discovery and functional analysis of small secreted proteins in plants

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Xiao-Li Hu ◽  
Haiwei Lu ◽  
Md Mahmudul Hassan ◽  
Jin Zhang ◽  
Guoliang Yuan ◽  
...  
Keyword(s):  
Plant Species ◽  
Protein Secretion ◽  
Functional Characterization ◽  
Secreted Proteins ◽  
Computational Algorithms ◽  
Biotic Stresses ◽  
Substantial Progress ◽  
Microbe Interactions ◽  
Secretion Pathways ◽  
Small Secreted Proteins

AbstractSmall secreted proteins (SSPs) are less than 250 amino acids in length and are actively transported out of cells through conventional protein secretion pathways or unconventional protein secretion pathways. In plants, SSPs have been found to play important roles in various processes, including plant growth and development, plant response to abiotic and biotic stresses, and beneficial plant–microbe interactions. Over the past 10 years, substantial progress has been made in the identification and functional characterization of SSPs in several plant species relevant to agriculture, bioenergy, and horticulture. Yet, there are potentially a lot of SSPs that have not been discovered in plant genomes, which is largely due to limitations of existing computational algorithms. Recent advances in genomics, transcriptomics, and proteomics research, as well as the development of new computational algorithms based on machine learning, provide unprecedented capabilities for genome-wide discovery of novel SSPs in plants. In this review, we summarize known SSPs and their functions in various plant species. Then we provide an update on the computational and experimental approaches that can be used to discover new SSPs. Finally, we discuss strategies for elucidating the biological functions of SSPs in plants.

scholarly journals The Fate of Lipid-Coated and Uncoated Fluorescent Nanodiamonds during Cell Division in Yeast

Nanomaterials ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 516
Author(s):  
Aryan Morita ◽  
Thamir Hamoh ◽  
Felipe P. Perona Martinez ◽  
Mayeul Chipaux ◽  
Alina Sigaeva ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Model Organism ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Daughter Cells ◽  
Diamond Particles ◽  
Fluorescent Nanodiamonds ◽  
Intracellular Fate

Fluorescent nanodiamonds are frequently used as biolabels. They have also recently been established for magnetic resonance and temperature sensing at the nanoscale level. To properly use them in cell biology, we first have to understand their intracellular fate. Here, we investigated, for the first time, what happens to diamond particles during and after cell division in yeast (Saccharomyces cerevisiae) cells. More concretely, our goal was to answer the question of whether nanodiamonds remain in the mother cells or end up in the daughter cells. Yeast cells are widely used as a model organism in aging and biotechnology research, and they are particularly interesting because their asymmetric cell division leads to morphologically different mother and daughter cells. Although yeast cells have a mechanism to prevent potentially harmful substances from entering the daughter cells, we found an increased number of diamond particles in daughter cells. Additionally, we found substantial excretion of particles, which has not been reported for mammalian cells. We also investigated what types of movement diamond particles undergo in the cells. Finally, we also compared bare nanodiamonds with lipid-coated diamonds, and there were no significant differences in respect to either movement or intracellular fate.

scholarly journals Analysis of Protein Localization and Secretory Pathway Function Using the Yeast Saccharomyces cerevisiae

2002 ◽  
Vol 1 (4) ◽  
pp. 173-192 ◽  
Author(s):  
Elizabeth Vallen
Keyword(s):  
Cell Biology ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Protein Localization ◽  
Chimeric Protein ◽  
Secretory Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Isolation And Characterization ◽  
Yeast Transformants ◽  
Histidinol Dehydrogenase

The isolation and characterization of mutants has been crucial in understanding a number of processes in the field of cell biology. In this exercise, students examine the effects of mutations in the secretory pathway on protein localization. Yeast strains deficient for synthesis of histidinol dehydrogenase are transformed with a plasmid encoding a chimeric protein. The chimera contains a signal sequence fused to histidinol dehydrogenase. A strain with a defect in the translocation of secretory proteins into the endoplasmic reticulum (ER) accumulates sufficient histidinol dehydrogenase in the cytoplasm to grow on media lacking histidine. In contrast, yeast proficient for secretion, or yeast with secretion defects later in the pathway, are unable to grow on media lacking histidine. Student analysis of the experimental yeast transformants and appropriate controls allows investigation into the effects of conditional defects in the secretory pathway on both cell viability and protein localization. The exercise is usually performed in a manner that allows students to execute a number of techniques common in molecular biology laboratories, including plasmid minipreps, restriction digestions, and Southern blots. Student understanding and enjoyment of the exercise was assessed by laboratory reports, oral and written examinations, and questionnaires. After completion of these experiments, students can describe the utility of protein fusions, the roles of mutant analysis in cell biology, and the steps taken by proteins transiting the secretory pathway.

scholarly journals Evaluation of Unconventional Protein Secretion by Saccharomyces cerevisiae and other Fungi

Cells ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 128 ◽  
Author(s):  
Natsuko Miura ◽  
Mitsuyoshi Ueda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Secretion ◽  
Model Organism ◽  
Proteome Analysis ◽  
Extracellular Vesicle ◽  
Fungal Species ◽  
Extracellular Proteins ◽  
Research Progress ◽  
Fungal Allergens ◽  
Secretion Pathways

Development of proteome analysis of extracellular proteins has revealed that a wide variety of proteins, including fungal allergens are present outside the cell. These secreted allergens often do not contain known secretion signal sequences. Recent research progress shows that some fungal allergens are secreted by unconventional secretion pathways, including autophagy- and extracellular-vesicle-dependent pathways. However, secretion pathways remain unknown for the majority of extracellular proteins. This review summarizes recent data on unconventional protein secretion in Saccharomyces cerevisiae and other fungi. Particularly, methods for evaluating unconventional protein secretion are proposed for fungal species, including S. cerevisiae, a popular model organism for investigating protein secretion pathways.

scholarly journals ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

2020 ◽  
Author(s):  
Jason C. Casler ◽  
Allison L. Zajac ◽  
Fernando M. Valbuena ◽  
Daniela Sparvoli ◽  
Okunola Jeyifous ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Signal Sequence ◽  
Model Organism ◽  
Kinetic Studies ◽  
Secretory Protein ◽  
Yeast Cells ◽  
Model Organisms ◽  
Cargo Protein ◽  
Simple Technology

AbstractMembrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid transport out of the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

scholarly journals Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

2021 ◽  
Vol 7 (2) ◽  
pp. 149
Author(s):  
Sarah-Maria Wege ◽  
Katharina Gejer ◽  
Fabienne Becker ◽  
Michael Bölker ◽  
Johannes Freitag ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Genomic Locus ◽  
Targeted Gene Deletion ◽  
Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

scholarly journals CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 692
Author(s):  
Sweta Talyan ◽  
Samantha Filipów ◽  
Michael Ignarski ◽  
Magdalena Smieszek ◽  
He Chen ◽  
...  
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
De Novo ◽  
Depth Information ◽  
Gene Products ◽  
Classical Analysis ◽  
Protein Coding ◽  
Bioinformatic Pipeline ◽  
Non Coding Rnas ◽  
Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

Oxidative protein folding in the mammalian endoplasmic reticulum

2004 ◽  
Vol 32 (5) ◽  
pp. 655-658 ◽  
Author(s):  
C.E. Jessop ◽  
S. Chakravarthi ◽  
R.H. Watkins ◽  
N.J. Bulleid
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Redox State ◽  
Structure And Function ◽  
Reduced Form ◽  
Secretory Proteins ◽  
Oxidative Protein Folding ◽  
Disulphide Bonds ◽  
And Function ◽  
Substrate Proteins

Native disulphide bonds are essential for the structure and function of many membrane and secretory proteins. Disulphide bonds are formed, reduced and isomerized in the endoplasmic reticulum of mammalian cells by a family of oxidoreductases, which includes protein disulphide isomerase (PDI), ERp57, ERp72, P5 and PDIR. This review will discuss how these enzymes are maintained in either an oxidized redox state that allows them to form disulphide bonds in substrate proteins or a reduced form that allows them to perform isomerization and reduction reactions, how these opposing pathways may co-exist within the same compartment and why so many oxidoreductases exist when PDI alone can perform all three of these functions.

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