L-Carnitine Tartrate Downregulates the ACE2 Receptor and Limits SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for one of the worst pandemics in modern history. Several prevention and treatment strategies have been designed and evaluated in recent months either through the repurposing of existing treatments or the development of new drugs and vaccines. In this study, we show that L-carnitine tartrate supplementation in humans and rodents led to significant decreases of key host dependency factors, notably angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and Furin, which are responsible for viral attachment, viral spike S-protein cleavage, and priming for viral fusion and entry. Interestingly, pre-treatment of Calu-3, human lung epithelial cells, with L-carnitine tartrate led to a significant and dose-dependent inhibition of the infection by SARS-CoV-2. Infection inhibition coincided with a significant decrease in ACE2 mRNA expression levels. These data suggest that L-carnitine tartrate should be tested with appropriate trials in humans for the possibility to limit SARS-CoV-2 infection.

NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.

Stepwise posttranslational processing of progrowth hormone-releasing hormone (proGHRH) polypeptide by furin and PC1.

Through a posttranslational processing mechanism, pro-growth hormone releasing hormone (proGHRH) gives rise to an amidated GHRH molecule, which in turn stimulates the synthesis and release of growth hormone. We have previously proposed a model for the biochemical processing of proGHRH [Nillni et al. (1999), Endocrinology 140, 5817-5827]. We demonstrated that the proGHRH peptide (10.5 kDa, 104 aa) is first processed to an 8.8 kDa intermediate form that is later cleaved to yield two products: the 5.2 kDa GHRH and the 3.6 kDa GHRH-RP. However, the proteolytic enzymes involved in this process are unknown. Therefore, in this study we determined which proconverting enzymes are involved in this process. We transfected different constructs in cell lines carrying different PC enzymes followed by analysis of the peptide products after metabolic labeling or Western blots. We found that in the absence of furin (LoVo cells) or CHO cells treated with BFA, only one moiety was observed, and that corresponds to the same electrophorectic mobility to the GHRH precursor. This finding strongly supports an initial role for furin in the processing of proGHRH. The results from transfections with preproGHRH alone or double or triple transfections with PC1 and PC2 in AtT-20, GH3, and GH4C1 cells indicated that PC1 is the primary enzyme involved in the generation of GHRH peptide from the 8.8 kDa intermediate form. We found that AtT-20 cells (high PC1, very low PC2) were able to generate GHRH. However, GH3 cells (high PC2, but not PC1) were able to process the 8.8 kDa peptide to GHRH only after the cotransfection with the PC1 enzyme. Transfections with preproGHRH-GFP and preproGHRH-V5 provided similar results in all the cell lines analyzed. These data support the hypothesis that proGHRH is initially cleave by furin at preproGHRH29-30, followed by a second cleavage at preproGHRH74 primarily by PC1 to generate GHRH and GHRH-RP peptides, respectively.

Altered processing of the neurotensin/neuromedin N precursor in PC2 knock down mice: a biochemical and immunohistochemical study.

Neurotensin (NT) and neuromedin N (NN) are generated by endoproteolytic cleavage of a common precursor molecule, pro-NT/NN. To gain insight into the role of prohormone convertases PC1, PC2, and PC7 in this process, we investigated the maturation of pro-NT/NN in the brain of PC7 (PC7-/-), PC2 (PC2-/-), and/or PC1 (PC1+/- and PC2-/-; PC1+/-) knock down mice. Inactivation of the PC7 gene was without effect, suggesting that this convertase is not involved in the processing of pro-NT/NN. By contrast, there was a 15% decrease in NT and a 50% decrease in NN levels, as measured by radioimmunoassay, in whole brain extracts from PC2 null as compared with wild type mice. Using immunohistochemistry, we found that this decrease in pro-NT/NN maturation products was uneven and that it was most pronounced in the medial preoptic area, lateral hypothalamus, and paraventricular hypothalamic nuclei. These results suggest that PC2 plays a critical role in the processing of pro-NT/NN in mouse brain and that its deficiency may be compensated to a regionally variable extent by other convertases. Previous data have suggested that PC1 might be subserving this role. However, there was no change in the maturation of pro-NT/NN in the brain of mice in which the PC1 gene had been partially inactivated, implying that complete PC1 knock down may be required for loss of function.