Biology subjects

Evagelidis, A.

Publications and source records attributed to Evagelidis, A..

4 recordsLinked to original sources

bioRxiv2022

Molecular interactions of PCSK9 with an inhibitory nanobody, CAP1 and HLA-C: functional regulation of LDLR levels

ObjectiveThe liver-derived circulating PCSK9 enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes. PCSK9 inhibition or silencing is presently used in clinics worldwide to reduce LDL-cholesterol, resulting in lower incidence of cardiovascular disease and possibly cancer/metastasis. The mechanism by which the PCSK9-LDLR complex is sorted to degradation compartments is not fully understood. We previously suggested that out of the three M1, M2 and M3 subdomains of the C-terminal Cys/His-rich-domain (CHRD) of PCSK9, only M2 is critical for the activity of extracellular of PCSK9 on cell surface LDLR. This likely implicates the binding of M2 to an unknown membrane-associated "protein X" that would escort the complex to endosomes/lysosomes for degradation. We reported that a nanobody P1.40 binds the M1 and M3 domains of the CHRD and inhibits the function of PCSK9. It was also reported that the cytosolic adenylyl cyclase-associated protein 1 (CAP1) could bind M1 and M3 subdomains and enhance the activity of PCSK9. In this study, we determined the 3-dimensional structure of the CHRD-P1.40 complex to understand the intricate interplay between P1.40, CAP1 and PCSK9 and how they regulate LDLR degradation. MethodsX-ray diffraction of the CHRD-P1.40 complex was analyzed with a 2.2 [A] resolution. The affinity and interaction of PCSK9 or CHRD with P1.40 or CAP1 was analyzed by atomic modeling, site-directed mutagenesis, bio-layer interferometry, expression in hepatic cell lines and immunocytochemistry to monitor LDLR degradation. The CHRD-P1.40 interaction was further analyzed by deep mutational scanning and binding assays to validate the role of predicted critical residues. Conformational changes and atomic models were obtained by small angle X-ray scattering (SAXS). ResultsWe demonstrate that PCSK9 exists in a closed or open conformation and that P1.40 favors the latter by binding key residues in the M1 and M3 subdomains of the CHRD. Our data show that CAP1 is well secreted by hepatic cells and binds extracellular PCSK9 at distinct residues in the M1 and M3 domains and in the acidic prodomain. CAP1 stabilizes the closed conformation of PCSK9 and prevents P1.40 binding. However, CAP1 siRNA only partially inhibited PCSK9 activity on the LDLR. By modeling the previously reported interaction between M2 and an R-X-E motif in HLA-C, we identified Glu567 and Arg549 as the critical M2 residues binding HLA-C. Amazingly, these two residues are also required for the PCSK9-induced LDLR degradation. ConclusionsThe present study reveals that CAP1 enhances the function of PCSK9, likely by twisting the protein into a closed configuration that exposes the M2 subdomain needed for targeting the PCSK9-LDLR complex to degradation compartments. We hypothesize that "protein X", which is expected to guide the LDLR-PCSK9-CAP1 complex to these compartments after endocytosis into clathrin-coated vesicles, is HLA-C or a similar MHC-I family member. This conclusion is supported by the PCSK9 natural loss-of-function Q554E and gain-of-function H553R M2 variants, whose consequences are anticipated by our modeling.

molecular biology↗

bioRxiv2021

Implications of Spike-glycoprotein processing at S1/S2 by Furin, at S2' by Furin and/or TMPRSS2 and shedding of ACE2: cell-to-cell fusion, cell entry and infectivity of SARS-CoV-2

Disclaimer StatementThe author has withdrawn this manuscript due to a duplicate posting of manuscript number 423106. Therefore, the author does not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author (Nabil G. Seidah at seidahn@ircm.qc.ca.

biochemistry↗

bioRxiv2021

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLRthat is shed by Furin

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote-carriers of ASGR1-deletions exhibited [~]34% lower risk of coronary artery disease and [~]10-14% non-HDL-cholesterol reduction. Since PCSK9 is a major degrader of LDLR, the regulation of LDLR and/or PCSK9 by ASGR1 was studied. Thus, we investigated the role of endogenous/overexpressed ASGR1 on LDLR degradation and functionality by Western-blot and immunofluorescence in HepG2 naive and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR and both interact with/enhance the degradation of the receptor independently. Such lack of cooperativity between PCSK9 and ASGR1 on LDLR expression was confirmed in livers of wild-type (WT) versus Pcsk9-/- mice. ASGR1-knockdown in HepG2 naive cells significantly increased total ([~]1.2-fold) and cell-surface ([~]4-fold) LDLR protein. In HepG2-PCSK9-knockout cells ASGR1-silencing led to [~]2-fold higher levels of LDLR protein and DiI-LDL uptake, associated with [~]9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 reduced primarily the immature non-O-glycosylated LDLR ([~]110 kDa), whereas the triple Gln240/Trp244/Glu253 Ala-mutant (loss of carbohydrate-binding) reduced the mature form of the LDLR ([~]150 kDa), suggesting that ASGR1 binds the LDLR in sugar-dependent and -independent fashion. Furin sheds ASGR1 at RKMK103{downarrow} into a secreted form, likely resulting in a loss-of-function on LDLR. LDLR is the first example of a liver-receptor ligand of ASGR1. Additionally, we demonstrate that lack of ASGR1 enhances LDLR levels and DiI-LDL incorporation, independently of PCSK9. Overall, silencing of ASGR1 and PCSK9 may lead to higher LDL-uptake by hepatocytes, thereby providing a novel approach to further reduce LDL-cholesterol.

biochemistry↗

bioRxiv2020

Furin cleaves SARS-CoV-2 spike-glycoprotein at S1/S2 and S2'for viral fusion/entry: indirect role for TMPRSS2

The spike (S)-protein of SARS-CoV-2 binds ACE2 and requires proteolytic "priming" at PRRAR685{downarrow} into S1 and S2 (cleavage at S1/S2), and "fusion-activation" at a S2 site for viral entry. In vitro, Furin cleaved peptides mimicking the S1/S2 cleavage site more efficiently than at the putative S2, whereas TMPRSS2 inefficiently cleaved both sites. In HeLa cells Furin-like enzymes mainly cleaved at S1/S2 during intracellular protein trafficking, and S2 processing by Furin at KPSKR815{downarrow} was strongly enhanced by ACE2, but not for the optimized S2 KRRKR815{downarrow} mutant (S2), whereas individual/double KR815AA mutants were retained in the endoplasmic reticulum. Pharmacological Furin-inhibitors (Boston Pharmaceuticals, BOS-inhibitors) effectively blocked endogenous S-protein processing in HeLa cells. Furthermore, we show using pseudotyped viruses that while entry by a "pH-dependent" endocytosis pathway in HEK293 cells did not require Furin processing at S1/S2, a "pH-independent" viral entry in lung-derived Calu-3 cells was sensitive to inhibitors of Furin (BOS) and TMPRSS2 (Camostat). Consistently, these inhibitors potently reduce infectious viral titer and cytopathic effects, an outcome enhanced when both compounds were combined. Quantitative analyses of cell-to-cell fusion and spike processing revealed the key importance of the Furin sites for syncytia formation. Our assays showed that TMPRSS2 enhances fusion and proteolysis at S2 in the absence of cleavage at S1/S2, an effect that is linked to ACE2 shedding by TMPRSS2. Overall, our results indicate that Furin and TMPRSS2 play synergistic roles in generating fusion-competent S-protein, and in promoting viral entry, supporting the combination of Furin and TMPRSS2 inhibitors as potent antivirals against SARS-CoV-2. IMPORTANCESARS-CoV-2 is the etiological agent of COVID-19 that resulted in >5 million deaths. The spike protein (S) of the virus directs infection of the lungs and other tissues by binding the angiotensin-converting enzyme 2 (ACE2) receptor. For effective infection, the S-protein is cleaved at two sites: S1/S2 and S2. Cleavage at S1/S2, induces a conformational change favoring the recognition of ACE2. The S2 cleavage is critical for cell-to-cell fusion and virus entry into host cells. Our study contributes to a better understanding of the dynamics of interaction between Furin and TMPRSS2 during SARS-CoV-2 entry and suggests that the combination of a non-toxic Furin inhibitor with a TMPRSS2 inhibitor could significantly reduce viral entry in lung cells, as evidenced by an average synergistic [~]95% reduction of viral infection. This represents a powerful novel antiviral approach to reduce viral spread in individuals infected by SARS-CoV-2 or future related coronaviruses.

biochemistry↗