Annexins-a family of proteins with distinctive tastes for cell signaling and membrane dynamics.

Annexins are cytosolic proteins with conserved three-dimensional structures that bind acidic phospholipids in cellular membranes at elevated Ca2+ levels. Through this they act as Ca2+-regulated membrane binding modules that organize membrane lipids, facilitating cellular membrane transport but also displaying extracellular activities. Recent discoveries highlight annexins as sensors and regulators of cellular and organismal stress, controlling inflammatory reactions in mammals, environmental stress in plants, and cellular responses to plasma membrane rupture. Here, we describe the role of annexins as Ca2+-regulated membrane binding modules that sense and respond to cellular stress and share our view on future research directions in the field.

P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2.

Upon proinflammatory challenges, endothelial cell surface presentation of the leukocyte receptor P-selectin, together with the stabilizing co-factor CD63, is needed for leukocyte capture and is mediated via demand-driven exocytosis from the Weibel-Palade bodies that fuse with the plasma membrane. We report that neutrophil recruitment to activated endothelium is significantly reduced in mice deficient for the endolysosomal cation channel TPC2 and in human primary endothelial cells with pharmacological TPC2 block. We observe less CD63 signal in whole-mount stainings of proinflammatory-activated cremaster muscles from TPC2 knockout mice. We find that TPC2 is activated and needed to ensure the transfer of CD63 from endolysosomes via Weibel-Palade bodies to the plasma membrane to retain P-selectin on the cell surface of human primary endothelial cells. Our findings establish TPC2 as a key element to leukocyte interaction with the endothelium and a potential pharmacological target in the control of inflammatory leukocyte recruitment.

Disruption of the Rab7-Dependent Final Common Pathway of Endosomal and Autophagic Processing Results in a Severe Podocytopathy.

SIGNIFICANCE STATEMENT: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates and complex morphology, such as podocytes. To improve our understanding on how disturbances of these trafficking pathways are linked to podocyte depletion and slit diaphragm (SD) injury, the authors explored the role of the small GTPase Rab7, which is linked to endosomal, lysosomal, and autophagic pathways, using as model systems mice and Drosophila with podocyte-specific or nephrocyte-specific loss of Rab7, and a human podocyte cell line depleted for Rab7. Their findings point to maturation and fusion events during endolysosomal and autophagic maturation as key processes for podocyte homeostasis and function and identify altered lysosomal pH values as a putative novel mechanism for podocytopathies. BACKGROUND: Endocytosis, recycling, and degradation of proteins are essential functions of mammalian cells, especially for terminally differentiated cells with limited regeneration rates, such as podocytes. How disturbances within these trafficking pathways may act as factors in proteinuric glomerular diseases is poorly understood. METHODS: To explore how disturbances in trafficking pathways may act as factors in proteinuric glomerular diseases, we focused on Rab7, a highly conserved GTPase that controls the homeostasis of late endolysosomal and autophagic processes. We generated mouse and Drosophila in vivo models lacking Rab7 exclusively in podocytes or nephrocytes, and performed histologic and ultrastructural analyses. To further investigate Rab7 function on lysosomal and autophagic structures, we used immortalized human cell lines depleted for Rab7. RESULTS: Depletion of Rab7 in mice, Drosophila , and immortalized human cell lines resulted in an accumulation of diverse vesicular structures resembling multivesicular bodies, autophagosomes, and autoendolysosomes. Mice lacking Rab7 developed a severe and lethal renal phenotype with early-onset proteinuria and global or focal segmental glomerulosclerosis, accompanied by an altered distribution of slit diaphragm proteins. Remarkably, structures resembling multivesicular bodies began forming within 2 weeks after birth, prior to the glomerular injuries. In Drosophila nephrocytes, Rab7 knockdown resulted in the accumulation of vesicles and reduced slit diaphragms. In vitro , Rab7 knockout led to similar enlarged vesicles and altered lysosomal pH values, accompanied by an accumulation of lysosomal marker proteins. CONCLUSIONS: Disruption within the final common pathway of endocytic and autophagic processes may be a novel and insufficiently understood mechanism regulating podocyte health and disease.

Importance of ACE2 for SARS-CoV-2 Infection of Kidney Cells.

In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SARS-CoV-2 into human kidney cells. First, the expression of the cellular receptor ACE2 was demonstrated to be decisive for viral SARS-CoV-2 cell entry in human AB8 podocytes, whereas the presence of the transmembrane protease serine 2 (TMPRSS2) was dispensable. Moreover, the ACE2 protein amount was well detectable by mass spectrometry analysis in human kidneys, while TMPRSS2 could be detected only in a few samples. Additionally, a negative correlation of the ACE2 protein abundance to male sex and elderly aged females in human kidney tissues was demonstrated in this work. Last, the possibility of a direct infection of kidney tubular renal structures by SARS-CoV-2 was demonstrated.

Pathophysiological Mechanisms of Cardiac Dysfunction in Transgenic Mice with Viral Myocarditis.

Viral myocarditis is pathologically associated with RNA viruses such as coxsackievirus B3 (CVB3), or more recently, with SARS-CoV-2, but despite intensive research, clinically proven treatment is limited. Here, by use of a transgenic mouse strain (TG) containing a CVB3ΔVP0 genome we unravel virus-mediated cardiac pathophysiological processes in vivo and in vitro. Cardiac function, pathologic ECG alterations, calcium homeostasis, intracellular organization and gene expression were significantly altered in transgenic mice. A marked alteration of mitochondrial structure and gene expression indicates mitochondrial impairment potentially contributing to cardiac contractile dysfunction. An extended picture on viral myocarditis emerges that may help to develop new treatment strategies and to counter cardiac failure.

Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier.

SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.

The MEK1/2 Inhibitor ATR-002 (Zapnometinib) Synergistically Potentiates the Antiviral Effect of Direct-Acting Anti-SARS-CoV-2 Drugs.

The coronavirus disease 2019 (COVID-19) represents a global public health burden. In addition to vaccination, safe and efficient antiviral treatment strategies to restrict the viral spread within the patient are urgently needed. An alternative approach to a single-drug therapy is the combinatory use of virus- and host-targeted antivirals, leading to a synergistic boost of the drugs' impact. In this study, we investigated the property of the MEK1/2 inhibitor ATR-002's (zapnometinib) ability to potentiate the effect of direct-acting antivirals (DAA) against SARS-CoV-2 on viral replication. Treatment combinations of ATR-002 with nucleoside inhibitors Molnupiravir and Remdesivir or 3C-like protease inhibitors Nirmatrelvir and Ritonavir, the ingredients of the drug Paxlovid, were examined in Calu-3 cells to evaluate the advantage of their combinatory use against a SARS-CoV-2 infection. Synergistic effects could be observed for all tested combinations of ATR-002 with DAAs, as calculated by four different reference models in a concentration range that was very well-tolerated by the cells. Our results show that ATR-002 has the potential to act synergistically in combination with direct-acting antivirals, allowing for a reduction in the effective concentrations of the individual drugs and reducing side effects.

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases.

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.

Repurposing Antifungals for Host-Directed Antiviral Therapy?

Because of their epidemic and pandemic potential, emerging viruses are a major threat to global healthcare systems. While vaccination is in general a straightforward approach to prevent viral infections, immunization can also cause escape mutants that hide from immune cell and antibody detection. Thus, other approaches than immunization are critical for the management and control of viral infections. Viruses are prone to mutations leading to the rapid emergence of resistant strains upon treatment with direct antivirals. In contrast to the direct interference with pathogen components, host-directed therapies aim to target host factors that are essential for the pathogenic replication cycle or to improve the host defense mechanisms, thus circumventing resistance. These relatively new approaches are often based on the repurposing of drugs which are already licensed for the treatment of other unrelated diseases. Here, we summarize what is known about the mechanisms and modes of action for a potential use of antifungals as repurposed host-directed anti-infectives for the therapeutic intervention to control viral infections.

The MEK1/2-inhibitor ATR-002 efficiently blocks SARS-CoV-2 propagation and alleviates pro-inflammatory cytokine/chemokine responses.

Coronavirus disease 2019 (COVID-19), the illness caused by a novel coronavirus now called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to more than 260 million confirmed infections and 5 million deaths to date. While vaccination is a powerful tool to control pandemic spread, medication to relieve COVID-19-associated symptoms and alleviate disease progression especially in high-risk patients is still lacking. In this study, we explore the suitability of the rapid accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway as a druggable target in the treatment of SARS-CoV-2 infections. We find that SARS-CoV-2 transiently activates Raf/MEK/ERK signaling in the very early infection phase and that ERK1/2 knockdown limits virus replication in cell culture models. We demonstrate that ATR-002, a specific inhibitor of the upstream MEK1/2 kinases which is currently evaluated in clinical trials as an anti-influenza drug, displays strong anti-SARS-CoV-2 activity in cell lines as well as in primary air-liquid-interphase epithelial cell (ALI) cultures, with a safe and selective treatment window. We also observe that ATR-002 treatment impairs the SARS-CoV-2-induced expression of pro-inflammatory cytokines, and thus might prevent COVID-19-associated hyperinflammation, a key player in COVID-19 progression. Thus, our data suggest that the Raf/MEK/ERK signaling cascade may represent a target for therapeutic intervention strategies against SARS-CoV-2 infections and that ATR-002 is a promising candidate for further drug evaluation.

Conserved cholesterol-related activities of Dispatched 1 drive Sonic hedgehog shedding from the cell membrane.

The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance-nodulation-division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.

Pharmacologically induced endolysosomal cholesterol imbalance through clinically licensed drugs itraconazole and fluoxetine impairs Ebola virus infection in vitro.

Ebola virus disease (EVD) is a severe and frequently lethal disease caused by Ebola virus (EBOV). The latest occasional EVD outbreak (2013-2016) in Western African, which was accompanied by a high fatality rate, showed the great potential of epidemic and pandemic spread. Antiviral therapies against EBOV are very limited, strain-dependent (only antibody therapies are available) and mostly restricted to symptomatic treatment, illustrating the urgent need for novel antiviral strategies. Thus, we evaluated the effect of the clinically widely used antifungal itraconazole and the antidepressant fluoxetine for a repurposing against EBOV infection. While itraconazole, similar to U18666A, directly binds to and inhibits the endosomal membrane protein Niemann-Pick C1 (NPC1), fluoxetine, which belongs to the structurally unrelated group of weakly basic, amphiphile so-called "functional inhibitors of acid sphingomyelinase" (FIASMA) indirectly acts on the lysosome-residing acid sphingomyelinase via enzyme detachment leading to subsequent lysosomal degradation. Both, the drug-induced endolysosomal cholesterol accumulation and the altered endolysosomal pH, might interfere with the fusion of viral and endolysosomal membrane, preventing infection with EBOV. We further provide evidence that cholesterol imbalance is a conserved cross-species mechanism to hamper EBOV infection. Thus, exploring the endolysosomal host-pathogen interface as a suitable antiviral treatment may offer a general strategy to combat EBOV infection.

Combination Therapy with Fluoxetine and the Nucleoside Analog GS-441524 Exerts Synergistic Antiviral Effects against Different SARS-CoV-2 Variants In Vitro.

The ongoing SARS-CoV-2 pandemic requires efficient and safe antiviral treatment strategies. Drug repurposing represents a fast and low-cost approach to the development of new medical treatment options. The direct antiviral agent remdesivir has been reported to exert antiviral activity against SARS-CoV-2. Whereas remdesivir only has a very short half-life time and a bioactivation, which relies on pro-drug activating enzymes, its plasma metabolite GS-441524 can be activated through various kinases including the adenosine kinase (ADK) that is moderately expressed in all tissues. The pharmacokinetics of GS-441524 argue for a suitable antiviral drug that can be given to patients with COVID-19. Here, we analyzed the antiviral property of a combined treatment with the remdesivir metabolite GS-441524 and the antidepressant fluoxetine in a polarized Calu-3 cell culture model against SARS-CoV-2. The combined treatment with GS-441524 and fluoxetine were well-tolerated and displayed synergistic antiviral effects against three circulating SARS-CoV-2 variants in vitro in the commonly used reference models for drug interaction. Thus, combinatory treatment with the virus-targeting GS-441524 and the host-directed drug fluoxetine might offer a suitable therapeutic treatment option for SARS-CoV-2 infections.

ANO5 in membrane repair - Status: "It's complicated".

ANO5/TMEM16E gene mutations are associated with myopathies. Two recent publications in the Journal of Cell Biology now both confirm that ANO5 deficiency results in defective plasma membrane repair and Ca2+ overload. But the big question is whether ANO5 acts at the plasma membrane or the endoplasmic reticulum.

Drug synergy of combinatory treatment with remdesivir and the repurposed drugs fluoxetine and itraconazole effectively impairs SARS-CoV-2 infection in vitro.

BACKGROUND AND PURPOSE: The SARS-COV-2 pandemic and the global spread of coronavirus disease 2019 (COVID-19) urgently call for efficient and safe antiviral treatment strategies. A straightforward approach to speed up drug development at lower costs is drug repurposing. Here, we investigated the therapeutic potential of targeting the interface of SARS CoV-2 with the host via repurposing of clinically licensed drugs and evaluated their use in combinatory treatments with virus- and host-directed drugs in vitro. EXPERIMENTAL APPROACH: We tested the antiviral potential of the antifungal itraconazole and the antidepressant fluoxetine on the production of infectious SARS-CoV-2 particles in the polarized Calu-3 cell culture model and evaluated the added benefit of a combinatory use of these host-directed drugs with the direct acting antiviral remdesivir, an inhibitor of viral RNA polymerase. KEY RESULTS: Drug treatments were well-tolerated and potently impaired viral replication. Importantly, both itraconazole-remdesivir and fluoxetine-remdesivir combinations inhibited the production of infectious SARS-CoV-2 particles > 90% and displayed synergistic effects, as determined in commonly used reference models for drug interaction. CONCLUSION AND IMPLICATIONS: Itraconazole-remdesivir and fluoxetine-remdesivir combinations are promising starting points for therapeutic options to control SARS-CoV-2 infection and severe progression of COVID-19.

Annexin Animal Models-From Fundamental Principles to Translational Research.

Routine manipulation of the mouse genome has become a landmark in biomedical research. Traits that are only associated with advanced developmental stages can now be investigated within a living organism, and the in vivo analysis of corresponding phenotypes and functions advances the translation into the clinical setting. The annexins, a family of closely related calcium (Ca2+)- and lipid-binding proteins, are found at various intra- and extracellular locations, and interact with a broad range of membrane lipids and proteins. Their impacts on cellular functions has been extensively assessed in vitro, yet annexin-deficient mouse models generally develop normally and do not display obvious phenotypes. Only in recent years, studies examining genetically modified annexin mouse models which were exposed to stress conditions mimicking human disease often revealed striking phenotypes. This review is the first comprehensive overview of annexin-related research using animal models and their exciting future use for relevant issues in biology and experimental medicine.

Special Issue "Recent Developments in Annexin Biology".

Discovered over 40 years ago, the annexin proteins were found to be a structurally conserved subgroup of Ca2+-binding proteins. While the initial research on annexins focused on their signature feature of Ca2+-dependent binding to membranes, over the years the biennial Annexin conference series has highlighted additional diversity in the functions attributed to the annexin family of proteins. The roles of these proteins now extend from basic science to biomedical research, and are being translated into the clinic. The research on annexins involves a global network of researchers, and the 10th biennial Annexin conference brought together over 80 researchers from ten European countries, USA, Brazil, Singapore, Japan and Australia for 3 days in September 2019. In this conference, the discussions focused on two distinct themes-the role of annexins in cellular organization and in health and disease. The articles published in this Special Issue cover these two main themes discussed at this conference, offering a glimpse into some of the notable findings in the field of annexin biology.

Characterization of the Atl-mediated staphylococcal internalization mechanism.

Staphylococcus aureus internalization by non-professional phagocytes is considered a main pathogenicity mechanism leading to chronic infections. The well-established mechanism of Staphylococcus aureus internalization is mediated by fibronectin (Fn)-binding proteins (FnBPs), Fn as a bridging molecule and the host cell α5ß1 integrin. We previously identified a novel alternative internalization mechanism in Staphylococcus aureus, which involves the major autolysin Atl and the host cell heat shock cognate protein 70 (Hsc70). Atl-dependent internalization is also employed by the coagulase-negative Staphylococcus epidermidis, where it might represent the major or even sole internalization mechanism, because of the lack of FnBP-homologous proteins. In this study, we aimed to further characterize the Atl-dependent staphylococcal internalization mechanism. We performed biomolecular interaction analysis (BIA) to quantify the adhesive properties of Atl and found multivalent and high affinity interactions of Atl with Fn and Hsc70. Confocal laser scanning microscopy (CLSM) and a flow-cytometric internalization assay in combination with different pharmacological inhibitors suggested an involvement of the α5ß1 integrin, Fn and Hsc70 and subsequent signaling events mediated by Src and phosphoinositide 3 (PI3) kinases in the Atl-dependent staphylococcal uptake by EA.hy 926 cells. Further characterization of the endocytic machinery implicated a role for clathrin-dependent receptor-mediated endocytosis involving actin cytoskeletal rearrangements and microtubules. In conclusion, Atl ubiquitous among staphylococcal species may substitute for the FnBPs ensuring low-level internalization via a mechanism that seems to share important features with the FnBP-mediated staphylococcal uptake potentially being the prerequisite for the development of therapy-resistant chronic infections by staphylococcal strains that lack FnBPs.

Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine.

The Coronavirus Disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Related Coronavirus 2 (SARS-CoV-2) is a global health emergency. As only very limited therapeutic options are clinically available, there is an urgent need for the rapid development of safe, effective, and globally available pharmaceuticals that inhibit SARS-CoV-2 entry and ameliorate COVID-19 severity. In this study, we explored the use of small compounds acting on the homeostasis of the endolysosomal host-pathogen interface, to fight SARS-CoV-2 infection. We find that fluoxetine, a widely used antidepressant and a functional inhibitor of acid sphingomyelinase (FIASMA), efficiently inhibited the entry and propagation of SARS-CoV-2 in the cell culture model without cytotoxic effects and also exerted potent antiviral activity against two currently circulating influenza A virus subtypes, an effect which was also observed upon treatment with the FIASMAs amiodarone and imipramine. Mechanistically, fluoxetine induced both impaired endolysosomal acidification and the accumulation of cholesterol within the endosomes. As the FIASMA group consists of a large number of small compounds that are well-tolerated and widely used for a broad range of clinical applications, exploring these licensed pharmaceuticals may offer a variety of promising antivirals for host-directed therapy to counteract enveloped viruses, including SARS-CoV-2.