Inhibition of Infectious HIV-1 Production by Rerouting the Cellular Furin Inhibitor Serpin B8.

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.

Variation of Proteolytic Cleavage Sites towards the N-Terminal End of the S2 Subunit of the Novel SARS-CoV-2 Omicron Sublineage BA.2.12.1.

The prevalence of novel SARS-CoV-2 variants is also accompanied by an increased turnover rate and additional cleavage sites at the positions necessary for priming the Spike (S) protein. Of these priming sites, the proteolytically sensitive polybasic sequence of the activation loop at the S1/S2 interface and the S2' location within the S2 subunit of the S protein are cleaved by furin and TMPRSS2, which are important for the infection of the target cell. Neutrophils, migrating to the site of infection, secrete serine proteases to fight against pathogens. The serine proteases encompass neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG), which can hydrolyze the peptide bond adjacent to the S1/S2 interface. SARS-CoV-2 might take the opportunity to hijack proteases from an immune response to support viral entry to the cell. The region near S704L within the S2 subunit, a novel amino acid substitution of SARS-CoV-2 Omicron sublineage BA.2.12.1, is located close to the S1/S2 interface. We found that NE, PR3, and CatG digested the peptide within this region; however, the S704L amino acid substitution altered cleavage sites for PR3. In conclusion, such an amino acid substitution modifies S2 antigen processing and might further impact the major histocompatibility complex (MHC) binding and T cell activation.

Camostat Does Not Inhibit the Proteolytic Activity of Neutrophil Serine Proteases.

Coronavirus disease 2019 (COVID-19) can lead to multi-organ failure influenced by comorbidities and age. Binding of the severe acute respiratory syndrome coronavirus 2 spike protein (SARS-CoV-2 S protein) to angiotensin-converting enzyme 2 (ACE2), along with proteolytic digestion of the S protein by furin and transmembrane protease serine subtype 2 (TMPRSS2), provokes internalization of SARS-CoV-2 into the host cell. Productive infection occurs through viral replication in the cytosol and cell-to-cell transmission. The catalytic activity of TMPRSS2 can be blocked by the trypsin-like serine protease inhibitor camostat, which impairs infection by SARS-CoV-2. At the site of infection, immune cells, such as neutrophils, infiltrate and become activated, releasing neutrophil serine proteases (NSPs), including cathepsin G (CatG), neutrophil elastase (NE), and proteinase 3 (PR3), which promote the mounting of a robust immune response. However, NSPs might be involved in infection and the severe outcome of COVID-19 since the uncontrolled proteolytic activity is responsible for many complications, including autoimmunity, chronic inflammatory disorders, cardiovascular diseases, and thrombosis. Here, we demonstrate that camostat does not inhibit the catalytic activity of CatG, NE, and PR3, indicating the need for additional selective serine protease inhibitors to reduce the risk of developing severe COVID-19.

Occurrence of a novel cleavage site for cathepsin G adjacent to the polybasic sequence within the proteolytically sensitive activation loop of the SARS-CoV-2 Omicron variant: The amino acid substitution N679K and P681H of the spike protein.

The serine proteases neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and neutrophil serine protease 4 (NSP4) are secreted by activated neutrophils as a part of the innate immune response against invading pathogens. However, these serine proteases might be adopted by viruses to mediate viral surface protein priming resulting in host cell entrance and productive infection. Indeed, NE and PR3 hydrolyze the scissile peptide bond within the proteolytically sensitive polybasic sequence of the activation loop of SARS-CoV-2 located at the S1/S2 interface of the Spike (S) protein; an amino acid motif which differs from SARS-CoV-1. The occurrence of novel SARS-CoV-2 variants and substitution of distinct amino acids at the polybasic sequence prompts serious concerns regarding increased transmissibility. We propose that a novel cleavage site by CatG of the Omicron variant and the increased substrate turnover of the Delta variant by furin within the polybasic sequence should be considered for increased transmission of SARS-CoV-2 variants.

Diisothiocyanate-Derived Mercapturic Acids Are a Promising Partner for Combination Therapies in Glioblastoma.

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma.

Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2+CD62L+Ly6Chi monocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

Hindrance of the Proteolytic Activity of Neutrophil-Derived Serine Proteases by Serine Protease Inhibitors as a Management of Cardiovascular Diseases and Chronic Inflammation.

During inflammation neutrophils become activated and segregate neutrophil serine proteases (NSPs) to the surrounding environment in order to support a natural immune defense. However, an excess of proteolytic activity of NSPs can cause many complications, such as cardiovascular diseases and chronic inflammatory disorders, which will be elucidated on a biochemical and immunological level. The application of selective serine protease inhibitors is the logical consequence in the management of the indicated comorbidities and will be summarized in this briefing.

Critical View of Novel Treatment Strategies for Glioblastoma: Failure and Success of Resistance Mechanisms by Glioblastoma Cells.

According to the invasive nature of glioblastoma, which is the most common form of malignant brain tumor, the standard care by surgery, chemo- and radiotherapy is particularly challenging. The presence of glioblastoma stem cells (GSCs) and the surrounding tumor microenvironment protects glioblastoma from recognition by the immune system. Conventional therapy concepts have failed to completely remove glioblastoma cells, which is one major drawback in clinical management of the disease. The use of small molecule inhibitors, immunomodulators, immunotherapy, including peptide and mRNA vaccines, and virotherapy came into focus for the treatment of glioblastoma. Although novel strategies underline the benefit for anti-tumor effectiveness, serious challenges need to be overcome to successfully manage tumorigenesis, indicating the significance of developing new strategies. Therefore, we provide insights into the application of different medications in combination to boost the host immune system to interfere with immune evasion of glioblastoma cells which are promising prerequisites for therapeutic approaches to treat glioblastoma patients.

What Animal Cancers teach us about Human Biology.

Cancers in animals present a large, underutilized reservoir of biomedical information with critical implication for human oncology and medicine in general. Discussing two distinct areas of tumour biology in non-human hosts, we highlight the importance of these findings for our current understanding of cancer, before proposing a coordinated strategy to harvest biomedical information from non-human resources and translate it into a clinical setting. First, infectious cancers that can be transmitted as allografts between individual hosts, have been identified in four distinct, unrelated groups, dogs, Tasmanian devils, Syrian hamsters and, surprisingly, marine bivalves. These malignancies might hold the key to improving our understanding of the interaction between tumour cell and immune system and, thus, allow us to devise novel treatment strategies that enhance anti-cancer immunosurveillance, as well as suggesting more effective organ and stem cell transplantation strategies. The existence of these malignancies also highlights the need for increased scrutiny when considering the existence of infectious cancers in humans. Second, it has long been understood that no linear relationship exists between the number of cells within an organism and the cancer incidence rate. To resolve what is known as Peto's Paradox, additional anticancer strategies within different species have to be postulated. These naturally occurring idiosyncrasies to avoid carcinogenesis represent novel potential therapeutic strategies.

Regulation of MHC I Molecules in Glioblastoma Cells and the Sensitizing of NK Cells.

Immunotherapy has been established as an important area in the therapy of malignant diseases. Immunogenicity sufficient for immune recognition and subsequent elimination can be bypassed by tumors through altered and/or reduced expression levels of major histocompatibility complex class I (MHC I) molecules. Natural killer (NK) cells can eliminate tumor cells in a MHC I antigen presentation-independent manner by an array of activating and inhibitory receptors, which are promising candidates for immunotherapy. Here we summarize the latest findings in recognizing and regulating MHC I molecules that affect NK cell surveillance of glioblastoma cells.

Neutrophil Elastase and Proteinase 3 Cleavage Sites Are Adjacent to the Polybasic Sequence within the Proteolytic Sensitive Activation Loop of the SARS-CoV-2 Spike Protein.

Serine proteases neutrophil elastase (NE), protease 3 (PR3), cathepsin G (CatG), and neutrophil serine protease 4 (NSP4) are released by activated neutrophils swarming around the place of pathogen invasion to provoke an immune response. However, uncontrolled proteolytic activity of proteases results in various human diseases, including cardiovascular diseases, thrombosis, and autoimmunity. In addition, proteases can be hijacked by several viruses to prime virus-derived surface proteins and evade immune detection by entering into the host cell. Indeed, porcine elastase increases the suitability of host cells to be infected by SARS-CoV-1. We compared the cleavage sites of human NE, PR3, and CatG as well as porcine-derived trypsin within the amino acid sequence of the proteolytic sensitive activation loop at the interface of S1/S2 of the spike protein (S protein) of SARS-CoV-1 as well as SARS-CoV-2. As a result, NE and PR3, but not CatG, hydrolyze the scissile peptide bond adjacent to the polybasic amino acid sequence of the S1/S2 interface of SARS-CoV-2, which is distinctive from SARS-CoV-1. These findings suggest that neutrophil-derived NE and PR3 participate in priming of the S1/S2 interface during an immune response.

Activity-Based Probes to Utilize the Proteolytic Activity of Cathepsin G in Biological Samples.

Neutrophils, migrating to the site of infection, are able to release serine proteases after being activated. These serine proteases comprise cathepsin G (CatG), neutrophil elastase protease 3 (PR3), and neutrophil serine protease 4 (NSP4). A disadvantage of the uncontrolled proteolytic activity of proteases is the outcome of various human diseases, including cardiovascular diseases, thrombosis, and autoimmune diseases. Activity-based probes (ABPs) are used to determine the proteolytic activity of proteases, containing a set of three essential elements: Warhead, recognition sequence, and the reporter tag for detection of the covalent enzyme activity-based probe complex. Here, we summarize the latest findings of ABP-mediated detection of proteases in both locations intracellularly and on the cell surface of cells, thereby focusing on CatG. Particularly, application of ABPs in regular flow cytometry, imaging flow cytometry, and mass cytometry by time-of-flight (CyTOF) approaches is advantageous when distinguishing between immune cell subsets. ABPs can be included in a vast panel of markers to detect proteolytic activity and determine whether proteases are properly regulated during medication. The use of ABPs as a detection tool opens the possibility to interfere with uncontrolled proteolytic activity of proteases by employing protease inhibitors.

Application of an Activity-Based Probe to Determine Proteolytic Activity of Cell Surface Cathepsin G by Mass Cytometry Data Acquisition.

During an immune response, cathepsin G (CatG) takes on the role of adaptive and innate immunity and the outcome depends on the localization of CatG. Soluble, cell surface-bound, or intracellular CatG is also responsible for pathophysiology conditions. We applied the activity-based probe MARS116-Bt to mass cytometry by time-of-flight to analyze CatG activity on the cell surface of immune cells. The phosphonate warhead of MARS116-Bt binds covalently to the serine amino acid residue S195 of the catalytic center and thereby CatG activity can be detected. This method contributes to observing the activation or inhibition status of cells during pathogenesis of diseases and enables accurate data acquisition from complex biological samples with a vast panel of cell subset markers in a single-cell resolution.

Influence of obesity on remodeling of lung tissue and organization of extracellular matrix after blunt thorax trauma.

BACKGROUND: Previously, it has been shown that obesity is a risk factor for recovery, regeneration, and tissue repair after blunt trauma and can affect the rate of muscle recovery and collagen deposition after trauma. To date, lung tissue regeneration and extracellular matrix regulation in obese mice after injury has not been investigated in detail yet. METHODS: This study uses an established blunt thorax trauma model to analyze morphological changes and alterations on gene and protein level in lean or obese (diet-induced obesity for 16 ± 1 week) male C57BL/6 J mice at various time-points after trauma induction (1 h, 6 h, 24 h, 72 h and 192 h). RESULTS: Morphological analysis after injury showed lung parenchyma damage at early time-points in both lean and obese mice. At later time-points a better regenerative capacity of lean mice was observed, since obese animals still exhibited alveoli collapse, wall thickness as well as remaining filled alveoli structures. Although lean mice showed significantly increased collagen and fibronectin gene levels, analysis of collagen deposition showed no difference based on colorimetric quantification of collagen and visual assessment of Sirius red staining. When investigating the organization of the ECM on gene level, a decreased response of obese mice after trauma regarding extracellular matrix composition and organization was detectable. Differences in the lung tissue between the diets regarding early responding MMPs (MMP8/9) and late responding MMPs (MMP2) could be observed on gene and protein level. Obese mice show differences in regulation of extracellular matrix components compared to normal weight mice, which results in a decreased total MMP activity in obese animals during the whole regeneration phase. Starting at 6 h post traumatic injury, lean mice show a 50% increase in total MMP activity compared to control animals, while MMP activity in obese mice drops to 50%. CONCLUSIONS: In conclusion, abnormal regulation of the levels of extracellular matrix genes in the lung may contribute to an aberrant regeneration after trauma induction with a delay of repair and pathological changes of the lung tissue in obese mice.

Cathepsin G and its Dichotomous Role in Modulating Levels of MHC Class I Molecules.

Cathepsin G (CatG) is involved in controlling numerous processes of the innate and adaptive immune system. These features include the proteolytic activity of CatG and play a pivotal role in alteration of chemokines as well as cytokines, clearance of exogenous and internalized pathogens, platelet activation, apoptosis, and antigen processing. This is in contrast to the capability of CatG acting in a proteolytic-independent manner due to the net charge of arginine residues in the CatG sequence which interferes with bacteria. CatG is a double-edged sword; CatG is also responsible in pathophysiological conditions, such as autoimmunity, chronic pulmonary diseases, HIV infection, tumor progression and metastasis, photo-aged human skin, Papillon-Lefèvre syndrome, and chronic inflammatory pain. Here, we summarize the latest findings for functional responsibilities of CatG in immunity, including bivalent regulation of major histocompatibility complex class I molecules, which underscore an additional novel role of CatG within the immune system.

Considering the Experimental use of Temozolomide in Glioblastoma Research.

Temozolomide (TMZ) currently remains the only chemotherapeutic component in the approved treatment scheme for Glioblastoma (GB), the most common primary brain tumour with a dismal patient's survival prognosis of only ~15 months. While frequently described as an alkylating agent that causes DNA damage and thus-ultimately-cell death, a recent debate has been initiated to re-evaluate the therapeutic role of TMZ in GB. Here, we discuss the experimental use of TMZ and highlight how it differs from its clinical role. Four areas could be identified in which the experimental data is particularly limited in its translational potential: 1. transferring clinical dosing and scheduling to an experimental system and vice versa; 2. the different use of (non-inert) solvent in clinic and laboratory; 3. the limitations of established GB cell lines which only poorly mimic GB tumours; and 4. the limitations of animal models lacking an immune response. Discussing these limitations in a broader biomedical context, we offer suggestions as to how to improve transferability of data. Finally, we highlight an underexplored function of TMZ in modulating the immune system, as an example of where the aforementioned limitations impede the progression of our knowledge.

Effects of yolkin on the immune response of mice and its plausible mechanism of action.

Yolkin is a product of proteolytic degradation of vitellogenin, a protein contained in eggs' yolk, with already described procognitive properties. Here, we investigated effects of yolkin on the humoral and cellular immune response in mice, phenotype of cells from lymphoid organs and function of innate immunity cells. In vitro studies included effects of yolkin on mitogen-induced thymocyte proliferation, percentage of CD19 cells in bone marrow cells culture, expression of signaling molecules in Jurkat cells, interleukin 2 receptor (IL-2R) subunits in WEHI 231 cells and susceptibility of these cells to anti-Ig-induced cell death. The results showed that repeatable i.p. injections of yolkin stimulated the humoral immune response to sheep red blood cells (SRBC) irrespective of the time of the treatment. On the other hand, yolkin inhibited contact sensitivity to oxazolone. Treatment of mice with yolkin diminished the percentage of double positive cells and increasing the content of single positive CD4+ and CD8+ cells in the thymus. At the same time an increase of percentage of CD19 + B cells in the spleen and mesenteric lymph nodes was observed. In addition, the protein, given i.p., diminished ex vivo ability to synthesize nitric oxide by resident, peritoneal macrophages, stimulated with lipopolisaccharide (LPS). In vitro studies showed that yolkin increased CD19+ cell content in bone marrow cell population. The protein also enhanced proliferation of thymocytes to concanavalin A and stimulated expression of MAP kinases in Jurkat cells. In WEHI 231 B cell line yolkin caused a loss of IL-2R gamma chain expression, correlated with an increased resistance of these cells to proapoptotic action of anti-Ig antibodies. In conclusion, this is a first demonstration of immunotropic properties of yolkin in in vitro and in vivo tests. The results provide evidence for induction of maturation and stimulatory signals in immature T and B cells by the protein, suggesting its potential role in the development of an embryo's immune system.

Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly.

Cathepsin G (CatG) is responsible for several distinct immune processes of adaptive and innate immunity depending on extra- or intracellular occurrence of CatG. Recently, we established a method to detect CatG activity at the cell surface of natural killer cells by using the activity-based probe MARS116-Bt in flow cytometry. MARS116-Bt consists of biotin, spacer, amino acid sequence, and a phosphonate warhead which binds covalently to the serine amino acid residue within the active center of CatG. Herein, MARS116 was conjugated to 5(6)-carboxyfluorescein (FAM) in order to limit non-specific signal-to-noise ratio generally resulting from binding of fluorescein-labelled avidin (avidin-FAM) to biotinylated, intracellular proteins; since MARS116-Bt is incubated with avidin-FAM in a second labelling step. MARS116-FAM was capable to detect intracellular CatG activity, in contrast to the control compound MARS116*-FAM which lacks the functional phosphonate warhead crucial for binding to the active-site of CatG and contains a carboxyl group instead. Furthermore, intracellular CatG activity was determined in CD4+ T cells, CD8+ T cells as well as in T regulatory cell (Treg) subsets. Thus, MARS116-FAM is a convenient activity-based probe to detect intracellular CatG activity in a flow cytometry approach.

Structure, regulation, and (patho-)physiological functions of the stress-induced protein kinase CK1 delta (CSNK1D).

Members of the highly conserved pleiotropic CK1 family of serine/threonine-specific kinases are tightly regulated in the cell and play crucial regulatory roles in multiple cellular processes from protozoa to human. Since their dysregulation as well as mutations within their coding regions contribute to the development of various different pathologies, including cancer and neurodegenerative diseases, they have become interesting new drug targets within the last decade. However, to develop optimized CK1 isoform-specific therapeutics in personalized therapy concepts, a detailed knowledge of the regulation and functions of the different CK1 isoforms, their various splice variants and orthologs is mandatory. In this review we will focus on the stress-induced CK1 isoform delta (CK1δ), thereby addressing its regulation, physiological functions, the consequences of its deregulation for the development and progression of diseases, and its potential as therapeutic drug target.