Low Density Lipoprotein Receptor-Related Protein 1 Dependent Endosomal Trapping and Recycling of Apolipoprotein E

BACKGROUND: Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling. PRINCIPAL FINDINGS: Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion. CONCLUSION: We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles

Genome Fragmentation Is Not Confined to the Peridinin Plastid in Dinoflagellates

When plastids are transferred between eukaryote lineages through series of endosymbiosis, their environment changes dramatically. Comparison of dinoflagellate plastids that originated from different algal groups has revealed convergent evolution, suggesting that the host environment mainly influences the evolution of the newly acquired organelle. Recently the genome from the anomalously pigmented dinoflagellate Karlodinium veneficum plastid was uncovered as a conventional chromosome. To determine if this haptophyte-derived plastid contains additional chromosomal fragments that resemble the mini-circles of the peridin-containing plastids, we have investigated its genome by in-depth sequencing using 454 pyrosequencing technology, PCR and clone library analysis. Sequence analyses show several genes with significantly higher copy numbers than present in the chromosome. These genes are most likely extrachromosomal fragments, and the ones with highest copy numbers include genes encoding the chaperone DnaK(Hsp70), the rubisco large subunit (rbcL), and two tRNAs (trnE and trnM). In addition, some photosystem genes such as psaB, psaA, psbB and psbD are overrepresented. Most of the dnaK and rbcL sequences are found as shortened or fragmented gene sequences, typically missing the 3′-terminal portion. Both dnaK and rbcL are associated with a common sequence element consisting of about 120 bp of highly conserved AT-rich sequence followed by a trnE gene, possibly serving as a control region. Decatenation assays and Southern blot analysis indicate that the extrachromosomal plastid sequences do not have the same organization or lengths as the minicircles of the peridinin dinoflagellates. The fragmentation of the haptophyte-derived plastid genome K. veneficum suggests that it is likely a sign of a host-driven process shaping the plastid genomes of dinoflagellates

Molecular Dynamics Simulation Studies to Probe the Impact of Oxidative Stress on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2

Color poster with text, images, charts, and graphs.The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as well as the human cell surface receptor angiotensin-converting enzyme II (ACE2) contain several cysteine residues. These cysteine residues exist either in the form of disulfide bridges (oxidized) or as thiols (reduced). The thiol-to-disulfide equilibrium is shifted to the right when there are excess reactive oxygen species (ROS) in the body, which is referred to as oxidative stress. It has been shown that certain preexisting conditions, associated with oxidative stress, such as diabetes, obesity, heart conditions, and age can put individuals at a higher risk of contracting COVID-19.  Therefore, we hypothesized that the oxidized state of these proteins may have an impact on the binding of the virus protein to the receptor. To test our hypothesis, we used molecular dynamics simulations to study the interacting residues at the binding interface of the complex formed by the receptor-binding domain of SARS-CoV-2 and the peptidase domain of ACE2. Four complexes of ACE2 and SARS-CoV-2 in different redox states were generated by either preserving the disulfides or reducing them to thiols. The preliminary data and findings of this study will be presented.National Conference on Undergraduate Research (NCUR); National Institute of Health; University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Role of a Conserved Disulfide on the Interactions Between Severe Acute Respiratory Syndrome Coronavirus 2 and Angiotensin-Converting Enzyme 2

Color poster with text, charts, and images.Coronaviruses are large, enveloped, positive strand RNA viruses capable of infecting a large array of mammalian and avian species that possess densely glycosylated spike-shaped proteins on their surfaces giving them the appearance of crowns under electron microscope, hence their name. The receptor binding domain (RBD) of the spike protein specifically recognizes and binds to the extracellular peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) with high affinity. There is some evidence to suggest that the entry of viral glycoprotein is affected by the thiol-disulfide balance on the cell surface and disrupting this balance can prevent the virus from being able to infect the host cell. Both the RBD of the spike protein and ACE2 contain several cystine residues, and the existence of several disulfide bridges within them has been established when the species are under oxidative stress. It has also been established that the complete reduction of these disulfide bridges to sulfhydryl groups completely impairs the ability of the RBD to bind to ACE2. However, it is still unknown how each individual disulfide bridge in these proteins impacts the binding. In this study, in a hope to gain an insight into a possible mechanism of disrupting the virus’s life cycle, the disulfide bridge between residues C344 and C361 in ACE2 were probed using molecular dynamics simulations. Results indicated that the removal of the investigated disulfide bridge is insufficient to disable binding between the proteins.University of Wisconsin--Eau Claire Office of Research and Sponsored Program

ApoE recycling is reduced in LRP1^−/− primary hepatocytes.

<p>LRP1<i>flox</i> mice were infected with AdEGFP or AdCre and three days after infection primary hepatocytes were isolated. Sixteen hours after seeding, the infection with AdCre resulted in a dramatic loss of LRP1 protein expression as determined by Western blotting (A) and indirect immunofluorescence (B–C). Pulse-chase experiments were performed by incubating LRP1-positive (LRP^+/+) and LRP1–negative (LRP^−/−) hepatocytes with Cy3-apoE-TRL for 60 min at 37°C. Cells were washed with heparin and incubated for additional 60 min at 37°C with media in the presence or absence (w/o) of 20 µg/ml HDL₃. Then supernatants were harvested and the amount of re-secreted Cy3-apoE was determined by SDS-PAGE and subsequent quantification as described above. HDL-induced apoE recycling was reduced in LRP1^−/− cells as seen by in-gel fluorescence of chase media (D). Quantification of 4 independent experiments ± S.E.M. revealed a strong reduction of HDL-induced apoE recycling in LRP1^−/− hepatocytes ± HDL (E).</p

LRP1-dependent sorting of apoE and RAP.

<p>Cell lysates from EGFP (lane 1 in A, B and C) or LRP1-EGFP (lane 2 in A, B and C) transfected cells were subjected to SDS-PAGE, and Western blotting was performed with antibodies against the 85 kDa subunit of LRP1 (A), the 515 kDa subunit of LRP1 (B) and the LDLR (C). The endogenous LRP1 precursor protein is cleaved by furin into 515 kDa and 85 kDa fragments (shown in lane 1 of A and B). The recombinant LRP-EGFP can be detected at approximately 600 kDa (lane 2 in A and B) and for the cleaved 85 kDa fragment fused to EGFP at 115 kDa (LRP1₈₅-EGFP; lane 2 in A). The overexpression of LRP1-EGFP expression slightly reduced the expression of LDLR (C). Western blotting using an antibody against beta actin verified equal protein loading. LRP1-EGFP transfected HEK293 cells were incubated with Cy3-apoE-TRL for 30 min. Confocal microscopy revealed strong colocalization of LRP1-EGFP and Cy3-apoE in endosomal compartments (D–E; see arrows in panel F; nuclei are stained with DAPI). High-magnification confocal live-cell microscopy of LRP1-EGFP expressing cells incubated with Cy3-RAP (G) or Cy3-apoE-TRL (H) revealed that RAP is still attached to the inner leaflet of the vesicular membrane and does not diffuse freely within the lumen (G, arrows and inlet). Additionally, RAP was detected in small, dense vesicles not containing LRP1 (G, arrowheads). In contrast, apoE was only present within LRP1-EGFP endosomes and evenly distributed inside the lumen (H, arrows). Bar is 5 (G–H) and 20 µm (D–F), respectively.</p

HDL stimulates the exit of apoE from LRP1- containing endosomes.

<p>HuH7 cells were incubated with Cy5-apoE-TRL for 20 minutes. ApoE recycling was then induced by Cy3-HDL. After 5 min (A–C) and 15 min (D–F), LRP1 (arrows) did no longer co-localize with Cy5-apoE which was instead found associated with Cy3-HDL (arrowheads; nuclei in blue). This was confirmed by high-magnification video-microscopy of LRP1-EGFP expressing HEK293 cells (G–I; due to short time-lapse between acquisition of red and blue channels, a minor signal offset due to endosomal movements was observed). Bar is 5 (G–I) and 20 µm (A–F), respectively.</p

Internalized RAP but not TRL-derived apoE is sorted towards lysosomes.

<p>Incubation of HuH7 cells with Cy5-apoE-TRL or Cy3-RAP was performed for 30 min at 37°C. Subsequent confocal immunofluorescence analysis revealed colocalization of LRP1 with apoE (A–B, see arrows in merged image C). Internalized Cy3-apoE did not appear within lysosomes as indicated by a counterstain with LAMP-1 (D–F). In contrast, after 30 min Cy3-RAP co-localized with LAMP-1 (G–H, see arrows in the merged image I), indicating lysosomal targeting of the LRP1 ligand RAP. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

TRL-derived apoE is internalized into peripheral endosomal compartments.

<p>Cy5-apoE-TRL and Cy3-RAP were prepared and protein labelling was analyzed by SDS-PAGE and subsequent in-gel fluorescence detection (A). Incubation of HuH7 cells without (B) and with Cy5-apoE-TRL (C) for 30 min at 37°C resulted in a punctuated endosomal pattern indicating receptor-mediated endocytosis. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p