Protein folding by the endoplasmic reticulum (ER) is physiologically
critical; its disruption causes ER stress and augments disease. ER
stress activates the unfolded protein response (UPR) to restore
...homeostasis. If stress persists, the UPR induces apoptotic cell death,
but the mechanisms remain elusive. Here, we report that unmitigated ER
stress promoted apoptosis through cell-autonomous, UPR-controlled
activation of death receptor 5 (DR5). ER stressors induced DR5
transcription via the UPR mediator CHOP; however, the UPR sensor IRE1α
transiently catalyzed DR5 mRNA decay, which allowed time for adaptation.
Persistent ER stress built up intracellular DR5 protein, driving
ligand-independent DR5 activation and apoptosis engagement via
caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress
and apoptotic cell fate.
Transition row metal ions are both essential and toxic to microorganisms. Zinc in excess has significant toxicity to bacteria, and host release of Zn(II) at mucosal surfaces is an important innate ...defence mechanism. However, the molecular mechanisms by which Zn(II) affords protection have not been defined. We show that in Streptococcus pneumoniae extracellular Zn(II) inhibits the acquisition of the essential metal Mn(II) by competing for binding to the solute binding protein PsaA. We show that, although Mn(II) is the high-affinity substrate for PsaA, Zn(II) can still bind, albeit with a difference in affinity of nearly two orders of magnitude. Despite the difference in metal ion affinities, high-resolution structures of PsaA in complex with Mn(II) or Zn(II) showed almost no difference. However, Zn(II)-PsaA is significantly more thermally stable than Mn(II)-PsaA, suggesting that Zn(II) binding may be irreversible. In vitro growth analyses show that extracellular Zn(II) is able to inhibit Mn(II) intracellular accumulation with little effect on intracellular Zn(II). The phenotype of S. pneumoniae grown at high Zn(II):Mn(II) ratios, i.e. induced Mn(II) starvation, closely mimicked a ΔpsaA mutant, which is unable to accumulate Mn(II). S. pneumoniae infection in vivo elicits massive elevation of the Zn(II):Mn(II) ratio and, in vitro, these Zn(II):Mn(II) ratios inhibited growth due to Mn(II) starvation, resulting in heightened sensitivity to oxidative stress and polymorphonuclear leucocyte killing. These results demonstrate that microbial susceptibility to Zn(II) toxicity is mediated by extracellular cation competition and that this can be harnessed by the innate immune response.
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify ...glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target.
Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target.
Glycointeractions in bacterial pathogenesis Poole, Jessica; Day, Christopher J; von Itzstein, Mark ...
Nature reviews. Microbiology,
07/2018, Letnik:
16, Številka:
7
Journal Article
Recenzirano
Many important interactions between bacterial pathogens and their hosts are highly specific binding events that involve host or pathogen carbohydrate structures (glycans). Glycan interactions can ...mediate adhesion, invasion and immune evasion and can act as receptors for toxins. Several bacterial pathogens can also enzymatically alter host glycans to reveal binding targets, degrade the host cell glycans or alter the function of host glycoproteins. In recent years, high-throughput screening technologies, such as lectin, glycan and mucin microarrays, have transformed the field by identifying new bacterial-host glycointeractions, which are crucial for colonization, persistence and disease. In this Review, we discuss interactions involving both host and bacterial glycans that have a role in bacterial pathogenesis. We also highlight recent technological advances that have illuminated the glycoscience of microbial pathogenesis.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signaling network known as the unfolded protein response (UPR). Here we characterize how ER stress ...and the UPR inhibit insulin signaling. We find that ER stress inhibits insulin signaling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesized insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signaling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible F
2E dimerization domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signaling events in the UPR, such as activation of the JNK mitogen-activated protein (MAP) kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signaling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesized insulin receptors in ER-stressed cells. Media: see text.
Protein transport into the mammalian endoplasmic reticulum (ER) is mediated by the heterotrimeric Sec61 channel. The signal recognition particle (SRP) and TRC systems and Sec62 have all been ...characterized as membrane-targeting components for small presecretory proteins, whereas Sec63 and the lumenal chaperone BiP act as auxiliary translocation components. Here, we report the transport requirements of two natural, small presecretory proteins and engineered variants using semipermeabilized human cells after the depletion of specific ER components. Our results suggest that hSnd2, Sec62, and SRP and TRC receptor each provide alternative targeting pathways for short secretory proteins and define rules of engagement for the actions of Sec63 and BiP during their membrane translocation. We find that the Sec62/Sec63 complex plus BiP can facilitate Sec61 channel opening, thereby allowing precursors that have weak signal peptides or other inhibitory features to translocate. A Sec61 inhibitor can mimic the effect of BiP depletion on Sec61 gating, suggesting that they both act at the same essential membrane translocation step.
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•Small human presecretory proteins use all known targeting routes to the Sec61 complex•Their insertion into Sec61 is selectively facilitated by BiP, Sec62, and Sec63•Selectivity is driven by weak signal peptides plus downstream inhibitory features•Cyclic heptadepsipeptides phenocopy the effect of BiP depletion on Sec61 gating
Protein transport into the human endoplasmic reticulum (ER) is mediated by the heterotrimeric Sec61 channel. Haßdenteufel et al. map the determinants for requirement of different targeting pathways and different auxiliary components of the Sec61 channel in ER import of short presecretory proteins. Different characteristics of precursor polypeptides dictate the engagement of each component.
In mammalian cells, one‐third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61 channel. While the Sec61 complex ...facilitates ER import of most precursor polypeptides, the Sec61‐associated Sec62/Sec63 complex supports ER import in a substrate‐specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63 complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified 22 novel Sec62/Sec63 substrates under these in vivo‐like conditions. As a common feature, those previously unknown substrates share signal peptides (SP) with comparatively longer but less hydrophobic hydrophobic region of SP and lower carboxy‐terminal region of SP (C‐region) polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly gating SP and a downstream translocation‐disruptive positively charged cluster of amino acid residues as decisive for the Sec62/Sec63 requirement. In the case of ERj3, these features were found to be responsible for an additional immunoglobulin heavy‐chain binding protein (BiP) requirement and to correlate with sensitivity toward the Sec61‐channel inhibitor CAM741. Thus, the human Sec62/Sec63 complex may support Sec61‐channel opening for precursor polypeptides with slowly gating SPs by direct interaction with the cytosolic amino‐terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER‐lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63‐linked polycystic liver disease.
Databases
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data (https://data.mendeley.com/datasets/6s5hn73jcv/2).
Sec61 is a membrane protein channel via which polypeptides are transported into the ER lumen or integrated into the membrane. The Sec61‐associated Sec62/Sec63 complex supports ER transport in a substate‐specific manner, but it is unclear how and which precursor polypeptides are recognised by this machinery. Here, Richard Zimmermann and co‐authors explored the mechanism of Sec62/Sec63‐driven ER import in human cells. They identified 36 precursors as Sec62/Sec63‐clients; 26 of which had signal peptides with longer but less hydrophobic H‐regions. Further analysis revealed that a slowly‐gating SP and the inhibitory effect of a positively charged cluster of amino acids are required for Sec62/Sec63 interaction.
Biosynthesis of insulin - critical to metabolic homeostasis - begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. ...Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes.
Streptococcus pneumoniae is a Gram-positive bacterial pathogen that colonizes the mucosal surfaces of the host nasopharynx and upper airway. Through a combination of virulence-factor activity and an ...ability to evade the early components of the host immune response, this organism can spread from the upper respiratory tract to the sterile regions of the lower respiratory tract, which leads to pneumonia. In this Review, we describe how S. pneumoniae uses its armamentarium of virulence factors to colonize the upper and lower respiratory tracts of the host and cause disease.
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