The fight against COVID-19: Striking a balance in the renin-angiotensin system.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells by interacting with membrane-bound angiotensin-converting enzyme 2 (ACE2), a vital element in the renin-angiotensin system (RAS), which regulates blood pressure, fluid balance, and cardiovascular functions. We herein evaluate existing evidence for the molecular alterations within the RAS pathway (e.g., ACE2 and angiotensin II) during SARS-CoV-2 infection and subsequent Coronavirus Disease 2019 (COVID-19). This includes reports regarding potential effect of RAS blockade (e.g., ACE inhibitors and angiotensin II receptor blockers) on ACE2 expression and clinical outcomes in patients with co-morbidities commonly treated with these agents. The collective evidence suggests a dual role for ACE2 in COVID-19, depending on the stage of infection and the coexisting diseases in individual patients. This information is further discussed with respect to potential therapeutic strategies targeting RAS for COVID-19 treatment.

Anthracycline-Induced Cardiotoxicity: Molecular Insights Obtained from Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs).

Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity. Here, we describe an assay platform by coupling hiPSC-CMs and impedance measurement, which allows real-time monitoring of cardiomyocyte cellular index, beating amplitude, and beating rate. Using this approach, we have performed comparative studies on a panel of four anthracycline drugs (doxorubicin, epirubicin, idarubicin, and daunorubicin) which share a high degree of structural similarity but are associated with distinct cardiotoxicity profiles and maximum cumulative dose limits. Notably, results from our hiPSC-CMs impedance model (dose-dependent responses and EC50 values) agree well with the recommended clinical dose limits for these drugs. Using time-lapse imaging and RNAseq, we found that the differences in anthracycline cardiotoxicity are closely linked to extent of cardiomyocyte uptake and magnitude of activation/inhibition of several cellular pathways such as death receptor signaling, ROS production, and dysregulation of calcium signaling. The results provide molecular insights into anthracycline cardiac interactions and offer a novel assay system to more robustly assess potential cardiotoxicity during drug development.

COVID-19 update: The race to therapeutic development.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents an unprecedented challenge to global public health. At the time of this review, COVID-19 has been diagnosed in over 40 million cases and associated with 1.1 million deaths worldwide. Current management strategies for COVID-19 are largely supportive, and while there are more than 2000 interventional clinical trials registered with the U.S. National Library of Medicine (clinicaltrials.gov), results that can clarify benefits and risks of candidate therapies are only gradually becoming available. We herein describe recent advances in understanding SARS-CoV-2 pathobiology and potential therapeutic targets that are involved in viral entry into host cells, viral spread in the body, and the subsequent COVID-19 progression. We highlight two major lines of therapeutic strategies for COVID-19 treatment: 1) repurposing the existing drugs for use in COVID-19 patients, such as antiviral medications (e.g., remdesivir) and immunomodulators (e.g., dexamethasone) which were previously approved for other disease conditions, and 2) novel biological products that are designed to target specific molecules that are involved in SARS-CoV-2 viral entry, including neutralizing antibodies against the spike protein of SARS-CoV-2, such as REGN-COV2 (an antibody cocktail), as well as recombinant human soluble ACE2 protein to counteract SARS-CoV-2 binding to the transmembrane ACE2 receptor in target cells. Finally, we discuss potential drug resistance mechanisms and provide thoughts regarding clinical trial design to address the diversity in COVID-19 clinical manifestation. Of note, preventive vaccines, cell and gene therapies are not within the scope of the current review.

Transcriptomic profiling reveals p53 as a key regulator of doxorubicin-induced cardiotoxicity.

Doxorubicin is an important anticancer drug in the clinic. Unfortunately, it causes cumulative and dose-dependent cardiotoxic side effects. As the population of cancer survivors who have been exposed to treatment continues to grow, there is increased interest in assessing the long-term cardiac effects of doxorubicin and understanding the underlying mechanisms at play. In this study, we investigated doxorubicin-induced transcriptomic changes using RNA-sequencing (RNAseq) and a cellular model comprised of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Analyses of predicted upstream regulators identified the p53 protein as a key regulator of transcriptomic changes induced by doxorubicin. Clustering and pathway analyses showed that increased death receptor (DR) expression and enrichment of the extrinsic apoptotic pathway are significantly associated with doxorubicin-induced cardiotoxicity. Increased expression of p53 and DRs were confirmed via immunoblotting. Our data pinpoints increased DR expression as an early transcriptomic indicator of cardiotoxicity, suggesting that DR expression might function as a predictive biomarker for cardiac damage.

Activity-based ubiquitin-protein probes reveal target protein specificity of deubiquitinating enzymes.

Ubiquitination is an essential eukaryotic post-translational modification that regulates various cellular processes. The removal of ubiquitin from its target protein is catalyzed by deubiquitinating enzymes (DUBs). Although it was proposed that many DUBs specifically interact and recognize ubiquitinated proteins as substrates, more direct evidence is needed to support this notion. Here we report protein-targeting activity-based DUB probes that allowed the identification of DUBs recognizing monoubiquitinated proliferating cell nuclear antigen (PCNA) in Saccharomyces cerevisiae. This new class of DUB probes contain a Michael acceptor as a warhead between ubiquitin and the target protein PCNA through a linkage that mimics the native isopeptide bond. We selected two known and biologically relevant ubiquitination sites on PCNA to generate the DUB probes. This allowed us to interrogate the site-specific deubiquitination of a target protein by DUBs. DUBs were profiled in yeast cell lysates using the two Ub-PCNA DUB probes in conjunction with two control probes that contain a noncleavable linkage but no warhead. We identified yeast DUBs through pulldown coupled with quantitative mass spectrometry analysis of the pulled down proteins. Our results showed that specific yeast DUBs recognize monoubiquitinated PCNA and corroborated previous genetic study. We also identified DUBs as potential new deubiquitinase of PCNA. Remarkably, identified DUBs clearly distinguish the different modification sites on PCNA, thus supporting a high level of DUB specificity beyond the target protein identity.

Cytokeratin 8/18 protects breast cancer cell lines from TRAIL-induced apoptosis.

TNF-related apoptosis inducing ligand (TRAIL) induces apoptosis by engaging its death receptors (DRs) 4 and/or 5 on targeted cells. Clinical attempts to stimulate this apoptotic pathway for cancer therapy, including the use of recombinant human TRAIL (rhTRAIL) or receptor agonistic antibodies, have been underway for over a decade. Unfortunately, these agents have only shown limited therapeutic effects due largely to tumor resistance arising from mechanisms yet to be defined. Here we show that intermediate filament proteins, keratin 8 and keratin 18 (K8/K18), negatively regulate TRAIL induced apoptosis. K8/K18 protein levels are consistently higher in TRAIL-resistant cells compared to TRAIL-sensitive cells in a panel of breast cancer cell lines. Blockade of K8 increased expression of DR5 on the surface of targeted cells and sensitized the cells to TRAIL-induced apoptosis. Conversely, ectopic expression of K8/K18 downregulated DR5 protein expression. K8/K18 appears to negatively regulate apoptosis signaling via DR5 in breast cancer cells. Our findings warrant additional studies to determine if K8/K18 could be a predictor of tumor resistance to DR5-targeted therapies.

RhoGDI deficiency induces constitutive activation of Rho GTPases and COX-2 pathways in association with breast cancer progression.

Rho GDP Dissociation Inhibitor (RhoGDI) is a key regulator of Rho GTPases. Here we report that loss of RhoGDI significantly accelerated xenograft tumor growth of MDA-MB-231 cells in animal models. At the molecular level, RhoGDI depletion resulted in constitutive activation of Rho GTPases, including RhoA, Cdc42, and Rac1. This was accompanied by Rho GTPase translocation from the cytosol to membrane compartments. Notably, COX-2 protein levels, mRNA expression, and biological activity were markedly increased in RhoGDI-deficient cells. The upregulated expression of COX-2 was directly associated with increased Rho GTPase activity. Further, we assessed the expression level of RhoGDI protein in breast tumor specimens (n = 165) by immunohistochemistry. We found that RhoGDI expression is higher in the early stages of breast cancer followed by a significant decrease in malignant tumors and metastatic lesions (p < 0.01). These data suggest that downregulation of RhoGDI could be a critical mechanism of breast tumor development, which may involve the hyperactivation of Rho GTPases and upregulation of COX-2 activity. Additional studies are warranted to evaluate the therapeutic potential of inhibiting Rho GTPases and COX-2 for treating breast cancers.

Spatial dynamics of TRAIL death receptors in cancer cells.

TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells without harming most normal cells. Currently, multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL (rhTRAIL) and agonistic antibodies that target death receptors (DRs) 4 or 5. It is encouraging that these products have shown a tolerated safety profile in early phase studies. However, their therapeutic potential is likely limited by the emergence of tumor drug resistance phenomena. Increasing evidence indicates that TRAIL DRs are deficient on the plasma membrane of some cancer cells despite their total protein expression. Notably, the lack of surface DR4/DR5 is sufficient to render cancers resistant to TRAIL-induced apoptosis, regardless of the status of other apoptosis signaling components. The current review highlights recent findings on the dynamic expression of TRAIL death receptors, including the regulatory roles of endocytosis, autophagy, and Ras GTPase-mediated signaling events. This information could aid in the identification of novel predictive biomarkers of tumor response as well as the development of combinational drugs to overcome or bypass tumor drug resistance to TRAIL receptor-targeted therapies.

Ricin detection: tracking active toxin.

Ricin is a plant toxin with high bioterrorism potential due to its natural abundance and potency in inducing cell death. Early detection of the active toxin is essential for developing appropriate countermeasures. Here we review concepts for designing ricin detection methods, including mechanism of action of the toxin, advantages and disadvantages of current detection assays, and perspectives on the future development of rapid and reliable methods for detecting ricin in environmental samples.

The use of a stably expressed FRET biosensor for determining the potency of cancer drugs.

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.

H-Ras regulation of TRAIL death receptor mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.

Transient kinetic analysis of USP2-catalyzed deubiquitination reveals a conformational rearrangement in the K48-linked diubiquitin substrate.

Deubiquitination has emerged as an essential regulatory mechanism of a number of cellular processes. An in-depth understanding of deubiquitinating enzyme (DUB) catalysis, particularly the mode of ubiquitin binding and the individual steps in the DUB catalytic turnover, is imperative for exploiting DUBs for therapeutic intervention. In this work, we present a transient kinetic study of USP2 in hydrolyzing a model substrate Ub-AMC and a physiological substrate K48-linked diubiquitin. We conducted stopped-flow fluorescence analyses of the binding of mono- and diubiquitin to an inactive USP2 mutant and unveiled interesting differences in the binding kinetics between the two substrates. While a simple one-step binding of monoubiquitin to USP2 was observed, a biphasic binding was evident for diubiquitin. We further followed the deubiquitination reaction of Ub-AMC and K48-linked IQF-diubiquitin by USP2 using stopped-flow florescence under a single-turnover condition. Global fitting of the reaction traces revealed differences in the microscopic rate constants between Ub-AMC and the physiological diubiquitin substrate. Our binding and single-turnover data support a conformational rearrangement of the diubiquitin substrate in USP2-catalyzed deubiquitination. This finding is significant given the recent finding that the K48-linked diubiquitin is dynamic in its conformation. Our results provide useful insights into the mechanism of how USP recognizes ubiquitin moieties in a chain structure, which is important for understanding USP catalysis and developing inhibitors against USPs.

Developing peptide-based multivalent antagonists of proliferating cell nuclear antigen and a fluorescence-based PCNA binding assay.

Proliferating cell nuclear antigen (PCNA) is a critical player in cell proliferation. It interacts with a myriad of cellular proteins in genomic DNA replication and cell cycle control. This makes PCNA an attractive target for developing antiproliferative therapeutics. Indeed, the binding of a human tumor suppressor protein, p21, to PCNA contributes to its antiproliferative effect in cells. In this work, we report a fluorescence polarization-based binding assay for determining the affinity between the p21 peptide and human PCNA. To improve the potency of the p21-based PCNA antagonist, we exploited the homotrimeric structure of PCNA and developed multivalent peptide-based PCNA antagonists. The di- and trivalent p21-based antagonists bind to PCNA with low nanomolar dissociation constant. Moreover, we show that the multivalent PCNA antagonists inhibited PCNA-dependent DNA synthesis in a human cell extract with improved avidity when compared with the monovalent p21 peptide. The fluorescence polarization assay holds promise for the discovery of potent small-molecule PCNA inhibitors given its ready adaptability to a high-throughput screening format.

Biochemical characterization of a multidomain deubiquitinating enzyme Ubp15 and the regulatory role of its terminal domains.

Deubiquitinating enzymes (DUBs) have emerged as essential players in a myriad of cellular processes, yet the regulation of DUB function remains largely unknown. While some DUBs rely on the formation of complex for regulation of enzymatic activity, many DUBs utilize interdomain interactions to regulate catalysis. Here we report the biochemical characterization of a multidomain deubiquitinating enzyme, Ubp15, from Saccharomyces cerevisiae. Steady-state kinetic investigation showed that Ubp15 is a highly active DUB. We identified active-site residues that are required for catalysis. We have also identified key residues on Ubp15 required for ubiquitin binding and catalysis. We further demonstrated that Ubp15's enzymatic activity is regulated by the N- and C-terminal domains that flank the catalytic core domain. Moreover, we demonstrated that Ubp15 physically interacts with a WD40 repeat-containing protein, Cdh1, by copurification experiments. Interestingly, unlike other DUBs that specifically interact with WD40 repeat-containing proteins, Cdh1 does not function in stimulating Ubp15's activity. The possible cellular function of Ubp15 in cell cycle regulation is discussed in view of the specific interaction between Ubp15 and Cdh1, an activator of the anaphase-promoting complex/cyclosome (APC/C).