Characterization of the enzymatic properties of human RNPEPL1/aminopeptidase Z.

It is now evident that the M1 family of aminopeptidases play important roles in many pathophysiological processes. Among them, the enzymatic properties of arginyl aminopeptidase-like 1 (RNPEPL1) are characterized only by its truncated form. No peptide substrate has been identified. To characterize the enzymatic properties of RNPEPL1 in more detail, the full-length protein was expressed in Escherichia coli and purified to homogeneity. The full-length RNPEPL1 showed rather restricted substrate specificity and basic amino acid preference towards synthetic substrates, which was different from the previously reported specificity characterized by the truncated form. Searching for peptide substrates, we found that several peptides, such as Met-enkephalin and kallidin, were cleaved. RNPEPL1 cleaved bradykinin to de-[Arg]-bradykinin despite the presence of proline at the P2'-position. The enzyme cleaved Met-enkephalin but not dynorphin A1-17. Similar to aminopeptidase B, the full-length RNPEPL1 showed basic amino acid preference towards both synthetic and peptide substrates. In addition to the unusual cleavage of bradykinin, this enzyme shows chain length-dependent cleavage of peptide substrates sharing N-terminal amino acid sequence. This is the first study to report the enzymatic properties of the full-length human RNPEPL1 as an aminopeptidase enzyme.

The dynamic nature of the coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia.

Once inhaled, SARS-CoV-2 particles enter respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). Understanding the nature of ACE2 within airway tissue has become a recent focus particularly in light of the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells and the air-liquid interface (ALI) model of differentiation. Using ALI tissue, three distinct transcript variants of ACE2 were identified. One transcript encodes the documented full-length ACE2 protein. The other two transcripts are unique truncated isoforms, that until recently had only been predicted to exist via sequence analysis software. Quantitative PCR revealed that all three transcript variants are expressed throughout differentiation of airway mucociliary epithelia. Immunofluorescence analysis of individual ACE2 protein isoforms exogenously expressed in cell-lines revealed similar abilities to localize in the plasma membrane and interact with the SARS CoV 2 spike receptor binding domain. Immunohistochemistry on differentiated ALI tissue using antibodies to either the N-term or C-term of ACE2 revealed both overlapping and distinct signals in cells, most notably only the ACE2 C-term antibody displayed plasma-membrane localization. We also demonstrate that ACE2 protein shedding is different in ALI Tissue compared to ACE2-transfected cell lines, and that ACE2 is released from both the apical and basal surfaces of ALI tissue. Together, our data highlights various facets of ACE2 transcripts and protein in airway mucociliary tissue that may represent variables which impact an individual's susceptibility to SARS-CoV-2 infection, or the severity of Covid-19.

Comprehensive in silico identification of impacts of ACE2 SNPs on COVID-19 susceptibility in different populations.

The COVID-19 pandemic emerges a reminder that wide spectrum discrepancy in response to SARS-CoV-2 infection and antiviral drugs among different populations might be due to their different ACE2 SNPs and/or miRNAs profile. ACE2 is the major component for SARS-CoV-2s' cell entry, and disruption of its 3D structure could influence virus-ACE2 interaction. In this study we aimed to investigate the consequence of 16,860 SNPs within ACE2 on its expression as well as protein folding, function, and stability by using several beneficial bioinformatics tools. Only 64 SNPs including 60 intronic, and 4 missense showed different frequencies among different populations. Two missense SNPs including rs149039346 and rs147311723 have been predicted to strongly influence the function and stability of ACE2. rs1514283 creates new acceptor splice site. Also, rs4646175 creates new donor and acceptor splice site. PolymiRTS, and miRSNPs have predicted that rs3746444, rs113808830, and rs3751304 showed a MAF > 0.001, and disrupted mRNA target sites or mRNA function. Finally, rs3746444 hsa-miR-499a-3p, rs113808830 hsa-miR-4532, rs3751304 hsa-miR-6763-3p and hsa-miR-26b-5p were strongly hybridized with ACE2 and might influence its function. Collectively, this study shed some light on fundamental roles of ACE2 SNPs for its interaction with COVID-19, and consequently susceptibility to virus. Therefore, different responses of patients with COVID-19 to ACE2 blocker drugs might be due to their unique ACE2 SNPs. We further discussed the impact of SNPs on miRNAs profile as a factor that may modulate drug response or susceptibility to COVID-19.

SARS-CoV-2 infection and smoking: What is the association? A brief review.

Susceptibility to severe illness from COVID-19 is anticipated to be associated with cigarette smoking as it aggravates the risk of cardiovascular and respiratory illness, including infections. This is particularly important with the advent of a new strain of coronaviruses, the severe acute respiratory syndrome coronavirus (SARS-CoV-2) that has led to the present pandemic, coronavirus disease 2019 (COVID-19). Although, the effects of smoking on COVID-19 are less described and controversial, we presume a link between smoking and COVID-19. Smoking has been shown to enhance the expression of the angiotensin-converting enzyme-2 (ACE-2) and transmembrane serine protease 2 (TMPRSS2) key entry genes utilized by SARS-CoV-2 to infect cells and induce a 'cytokine storm', which further increases the severity of COVID-19 clinical course. Nevertheless, the impact of smoking on ACE-2 and TMPRSS2 receptors expression remains paradoxical. Thus, further research is necessary to unravel the association between smoking and COVID-19 and to pursue the development of potential novel therapies that are able to constrain the morbidity and mortality provoked by this infectious disease. Herein we present a brief overview of the current knowledge on the correlation between smoking and the expression of SARS-CoV-2 key entry genes, clinical manifestations, and disease progression.

Activation of Nrf2/HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress.

Introduction: Recently, Nrf2/HO-1 has received extensive attention as the main regulatory pathway of intracellular defense against oxidative stress and is considered an ideal target for alleviating endothelial cell (EC) injury. Objectives: This paper aimed to summarized the natural monomers/extracts that potentially exert protective effects against oxidative stress in ECs. Methods: A literature search was carried out regarding our topic with the keywords of "atherosclerosis" or "Nrf2/HO-1" or "vascular endothelial cells" or "oxidative stress" or "Herbal medicine" or "natural products" or "natural extracts" or "natural compounds" or "traditional Chinese medicines" based on classic books of herbal medicine and scientific databases including Pubmed, SciFinder, Scopus, the Web of Science, GoogleScholar, BaiduScholar, and others. Then, we analyzed the possible molecular mechanisms for different types of natural compounds in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress. In addition, perspectives for possible future studies are discussed. Results: These agents with protective effects against oxidative stress in ECs mainly include phenylpropanoids, flavonoids, terpenoids, and alkaloids. Most of these agents alleviate cell apoptosis in ECs due to oxidative stress, and the mechanisms are related to Nrf2/HO-1 signaling activation. However, despite continued progress in research on various aspects of natural agents exerting protective effects against EC injury by activating Nrf2/HO-1 signaling, the development of new drugs for the treatment of atherosclerosis (AS) and other CVDs based on these agents will require more detailed preclinical and clinical studies. Conclusion: Our present paper provides updated information of natural agents with protective activities on ECs against oxidative stress by activating Nrf2/HO-1. We hope this review will provide some directions for the further development of novel candidate drugs from natural agents for the treatment of AS and other CVDs.

Adipose tissue (P)RR regulates insulin sensitivity, fat mass and body weight.

OBJECTIVE: We previously demonstrated that the handle-region peptide, a prorenin/renin receptor [(P)RR] blocker, reduces body weight and fat mass and may improve insulin sensitivity in high-fat fed mice. We hypothesized that knocking out the adipose tissue (P)RR gene would prevent weight gain and insulin resistance. METHODS: An adipose tissue-specific (P)RR knockout (KO) mouse was created by Cre-loxP technology using AP2-Cre recombinase mice. Because the (P)RR gene is located on the X chromosome, hemizygous males were complete KO and had a more pronounced phenotype on a normal diet (ND) diet compared to heterozygous KO females. Therefore, we challenged the female mice with a high-fat diet (HFD) to uncover certain phenotypes. Mice were maintained on either diet for 9 weeks. RESULTS: KO mice had lower body weights compared to wild-types (WT). Only hemizygous male KO mice presented with lower total fat mass, higher total lean mass as well as smaller adipocytes compared to WT mice. Although food intake was similar between genotypes, locomotor activity during the active period was increased in both male and female KO mice. Interestingly, only male KO mice had increased O2 consumption and CO2 production during the entire 24-hour period, suggesting an increased basal metabolic rate. Although glycemia during a glucose tolerance test was similar, KO males as well as HFD-fed females had lower plasma insulin and C-peptide levels compared to WT mice, suggesting improved insulin sensitivity. Remarkably, all KO animals exhibited higher circulating adiponectin levels, suggesting that this phenotype can occur even in the absence of a significant reduction in adipose tissue weight, as observed in females and, thus, may be a specific effect related to the (P)RR. CONCLUSIONS: (P)RR may be an important therapeutic target for the treatment of obesity and its associated complications such as type 2 diabetes.