Circulating mortalin in blood and activation of the alternative complement pathway as risk indicators in COVID-19 infection.

Background: Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70. It protects cells from various types of damages and from senescence. Our goal was to determine whether COVID-19 patients have circulating mortalin in their blood and to assess its prognostic value in anticipating disease severity. Methods: Mortalin was determined by ELISA in the sera of 83 COVID-19 patients enrolled in the study. Patients were categorized into 4 groups: critical patients who died (FATAL) or required intensive care and survived (ICU), patients of mild severity (hospitalized but not critical) who required nasal oxygen support (HOSP+O2), and patients who did not need oxygen therapy (HOSP). Results: The mortalin concentration in the serum of all COVID-19 patients in the cohort was 194-2324 pg/mL. A comparison of the mortalin levels by peak severity among the various patient groups showed a highly significant difference between the HOSP and FATAL groups and a significant difference between the HOSP and the ICU groups. COVID-19 patients who eventually failed to survive had at hospitalization a markedly higher level of mortalin in their sera. Cox regression analysis revealed a high mortality hazard (HR=3.96, p<0.01) in patients with high mortalin circulating levels (above the median, ≥651 pg/mL). This was confirmed in survival curve analysis (Kaplan-Meier; p=0.0032, log-rank test). Mortalin remained an independent predictor of mortality even after adjusting for age and sex or various complement activation products. Complement activation data collected in an earlier study in the same cohort was compared regarding the mortalin levels. Patients with higher circulating mortalin levels also had higher levels of complement C3a but reduced levels of properdin. Discussion: This is the first report on circulating mortalin in COVID-19 patients. Higher mortalin levels were associated with more severe illnesses and a higher risk of death. We claim that quantifying the blood levels of mortalin and activated complement proteins will provide important information on the prognosis of COVID-19 patients and will serve as a useful tool for guiding their clinical management and treatment.

Cell-Penetrating Peptide-Based Delivery of Macromolecular Drugs: Development, Strategies, and Progress.

Cell-penetrating peptides (CPPs), developed for more than 30 years, are still being extensively studied due to their excellent delivery performance. Compared with other delivery vehicles, CPPs hold promise for delivering different types of drugs. Here, we review the development process of CPPs and summarize the composition and classification of the CPP-based delivery systems, cellular uptake mechanisms, influencing factors, and biological barriers. We also summarize the optimization routes of CPP-based macromolecular drug delivery from stability and targeting perspectives. Strategies for enhanced endosomal escape, which prolong its half-life in blood, improved targeting efficiency and stimuli-responsive design are comprehensively summarized for CPP-based macromolecule delivery. Finally, after concluding the clinical trials of CPP-based drug delivery systems, we extracted the necessary conditions for a successful CPP-based delivery system. This review provides the latest framework for the CPP-based delivery of macromolecular drugs and summarizes the optimized strategies to improve delivery efficiency.

Mortalin peptides exert antitumor activities and act as adjuvants to antibody-mediated complement-dependent cytotoxicity.

Cancer cells have developed numerous strategies to maintain their proliferative capacity and to withstand different kinds of stress. The mitochondrial stress­70 protein named glucose regulated protein 75 (GRP75), also known as mortalin, is an intriguing cancer pro­survival factor. It is constitutively expressed in normal tissues but is upregulated in many tumors, and was shown to be a cancer prognostic biomarker. Mortalin is an inhibitor of complement­dependent cytotoxicity (CDC) and may therefore protect cells from antibody­based immunotherapy. To target mortalin for cancer therapy, our laboratory designed several mortalin mimetic peptides with sequences predicted to be involved in mortalin binding to its client proteins. The peptides were synthesized with a C­terminal transactivator of transcription sequence. By using cell death methodologies, the mechanism of action of the mortalin mimetic peptides on cancer cells was studied. Two peptides in particular, Mot­P2 and Mot­P7, were found to be highly toxic to lymphoma and ovarian, breast and prostate carcinoma cells. The analysis of their mode of action revealed that they may induce, within minutes, plasma membrane perturbations and mitochondrial stress. Furthermore, Mot­P2 and Mot­P7 activated necrotic cell death, leading to plasma membrane perforation, mitochondrial inner membrane depolarization and decrease in ATP level. In addition, Mot­P7, but not Mot­P2, required extracellular calcium ions to fully mediate cell death and was partially inhibited by plasma membrane cholesterol. At sub­toxic concentrations, the two peptides moderately inhibited cancer cell proliferation and blocked cell cycle at G2/M. Both peptides may bind intracellularly to mortalin and/or a mortalin­binding protein, hence knocking down mortalin expression reduced cell death. Combining treatment with Mot­P2 or Mot­P7 and CDC resulted in increased cell death. This study identified highly cytotoxic mortalin mimetic peptides that may be used as monotherapy or combined with complement­activating antibody therapy to target mortalin for precision cancer therapy.

MicroRNAs Affect Complement Regulator Expression and Mitochondrial Activity to Modulate Cell Resistance to Complement-Dependent Cytotoxicity.

MicroRNAs (miR) are small RNA molecules that shape the cell transcriptome and proteome through regulation of mRNA stability and translation. Here, we examined their function as determinants of cell resistance to complement-dependent cytotoxicity (CDC). To achieve this goal, we compared the expression of microRNAs between complement-resistant and -sensitive K562 leukemia, Raji lymphoma, and HCT-116 colorectal carcinoma cells. Global microRNA array analysis identified miR-150, miR-328, and miR-616 as regulators of CDC resistance. Inhibition of miR-150 reduced resistance, whereas inhibition of miR-328 or miR-616 enhanced cell resistance. Treatment of K562 cells with a sublytic dose of complement was shown to rapidly increase miR-150, miR-328, and miR-616 expression. Protein targets of these microRNAs were analyzed in K562 cells by mass spectrometry-based proteomics. Expression of the complement membrane regulatory proteins CD46 and CD59 was significantly enhanced after inhibition of miR-328 and miR-616. Enrichment of proteins of mitochondria, known target organelles in CDC, was observed after miR-150, miR-328, and miR-616 inhibition. In conclusion, miR-150, miR-328, and miR-616 regulate cell resistance to CDC by modifying the expression of the membrane complement regulators CD46 and CD59 and the response of the mitochondria to complement lytic attack. These microRNAs may be considered targets for intervention in complement-associated diseases and in anticancer, complement-based therapy.

Complement C5b-9 and Cancer: Mechanisms of Cell Damage, Cancer Counteractions, and Approaches for Intervention.

The interactions of cancer cells with components of the complement system are highly complex, leading to an outcome that is either favorable or detrimental to cancer cells. Currently, we perceive only the "tip of the iceberg" of these interactions. In this review, we focus on the complement terminal C5b-9 complex, known also as the complement membrane attack complex (MAC) and discuss the complexity of its interaction with cancer cells, starting with a discussion of its proposed mode of action in mediating cell death, and continuing with a portrayal of the strategies of evasion exhibited by cancer cells, and closing with a proposal of treatment approaches targeted at evasion strategies. Upon intense complement activation and membrane insertion of sufficient C5b-9 complexes, the afflicted cells undergo regulated necrotic cell death with characteristic damage to intracellular organelles, including mitochondria, and perforation of the plasma membrane. Several pro-lytic factors have been proposed, including elevated intracellular calcium ion concentrations and activated JNK, Bid, RIPK1, RIPK3, and MLKL; however, further research is required to fully characterize the effective cell death signals activated by the C5b-9 complexes. Cancer cells over-express a multitude of protective measures which either block complement activation, thus reducing the number of membrane-inserted C5b-9 complexes, or facilitate the elimination of C5b-9 from the cell surface. Concomitantly, cancer cells activate several protective pathways that counteract the death signals. Blockage of complement activation is mediated by the complement membrane regulatory proteins CD46, CD55, and CD59 and by soluble complement regulators, by proteases that cleave complement proteins and by protein kinases, like CK2, which phosphorylate complement proteins. C5b-9 elimination and inhibition of cell death signals are mediated by caveolin and dynamin, by Hsp70 and Hsp90, by the mitochondrial stress protein mortalin, and by the protein kinases PKC and ERK. It is conceivable that various cancers and cancers at different stages of development will utilize distinct patterns of these and other MAC resistance strategies. In order to enhance the impact of antibody-based therapy on cancer, novel precise reagents that block the most effective protective strategies will have to be designed and applied as adjuvants to the therapeutic antibodies.

Receptor-Interacting Protein Kinases 1 and 3, and Mixed Lineage Kinase Domain-Like Protein Are Activated by Sublytic Complement and Participate in Complement-Dependent Cytotoxicity.

The complement system participates in the pathogenesis of many diseases. Complement activation produces several active protein complexes and peptides, including the terminal C5b-9 complexes. It was reported that C5b-9 complexes insert into the plasma membrane and cause membrane perturbation, intracellular calcium surge, metabolic depletion, and osmotic lysis. Previously, we showed that complement-dependent cytotoxicity (CDC) is regulated by JNK and Bid. Here, we demonstrate that three mediators in TNFα-induced necroptosis (regulated necrosis), the receptor-interacting protein kinases, receptor-interacting protein kinase 1 (RIPK1) and receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like protein (MLKL), are activated by complement and contribute to CDC. Cell treatment with necrostatin-1 (Nec-1), a RIPK1 inhibitor, GSK'872, a RIPK3 inhibitor, or necrosulfonamide and GW806742X, MLKL inhibitors, restrain CDC. These findings were confirmed by using specific siRNAs targeting the synthesis of these proteins. Mouse fibroblasts lacking RIPK3 or MLKL were found to be less sensitive to C5b-9 than were wild-type (WT) fibroblasts. Enhanced CDC was achieved by RIPK1 or RIPK3 overexpression but not by the overexpression of a RHIM-RIPK1 mutant nor by a kinase-dead RIPK3 mutant. Nec-1 reduces the CDC of WT but not of RIPK3-knockout fibroblasts. Cells treated with a sublytic dose of complement exhibit co-localization of RIPK3 with RIPK1 in the cytoplasm and co-localization of RIPK3 and MLKL with C5b-9 at the plasma membrane. Data supporting cooperation among the RIP kinases, MLKL, JNK, and Bid in CDC are presented. These results provide a deeper insight into the cell death process activated by complement and identify potential points of cross talk between complement and other inducers of inflammation and regulated necrosis.

Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9.

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.

Circulating mitochondrial stress 70 protein/mortalin and cytosolic Hsp70 in blood: Risk indicators in colorectal cancer.

Mitochondrial mortalin and cytosolic Hsp70 are essential chaperones overexpressed in cancer cells. Our goals were to reproduce our earlier findings of elevated circulating levels of mortalin and Hsp70 in colorectal cancer (CRC) patients with a larger patient cohort, to compare death risk assessment of mortalin, Hsp70, CEA and C19-9 and to assess their prognostic value in various CRC stages. Mortalin, Hsp70, CEA and CA19-9 levels were determined in sera of 235 CRC patients enrolled in the study and followed up 5 years after surgery. Association between their concentrations and patients' survival was analyzed by Kaplan-Meier estimator and subjected to Cox Proportional hazards analysis. Serum level of mortalin was independent of that of Hsp70, CEA and CA19-9, whereas Hsp70 level weakly correlated with CEA and CA19-9 levels. Improved short-term survival was found in early or advanced disease stages associated with lower mortalin and Hsp70 levels. Cox regression analysis showed a high mortality hazard (HR = 3.7, p < 0.001) in patients with both high mortalin and Hsp70 circulating levels. Multivariate analysis showed that high mortalin and Hsp70 significantly enhances risk score over a baseline model of age, number of affected lymph nodes, CEA, CA19-9, disease stage and perioperative therapy. Analysis of mortalin and Hsp70 in CRC patients' sera adds a high prognostic value to TNM stage and to CEA and CA19-9 and identifies patients with lower or higher survival probability in all CRC stages. Determination of mortalin and Hsp70 in blood could be a useful additive prognostic tool in guiding clinical management of patients.

Complement triggers relocation of Mortalin/GRP75 from mitochondria to the plasma membrane.

Mortalin/GRP75 is a ubiquitously expressed mitochondrial chaperon that is overexpressed in cancer. Mortalin protects cells from complement-dependent cytotoxicity (CDC) and facilitates elimination of the complement C5b-9 complexes from the cell surface. We performed a nanoscopical study aimed at imaging the distribution of the C5b-9 complexes in the plasma membrane and the postulated relocation of mortalin from the mitochondria to the plasma membrane. To gain a resolution of 35nm, the locations of the C5b-9 complex and mortalin were imaged with a STED (Stimulated Emission Depletion) microscope at sub-diffraction resolution. Early changes in the spatial distribution of the C5b-9 on the cell surface are described. Juxtaposition of the labeled mortalin and C5b-9 at the plasma membrane region within minutes after complement attack is evident. Microscopical analysis of the distribution of mortalin in the vicinity of the mitochondria of complement-treated cells shows a more diffused pattern relative to control cells, proposing exit of mortalin from the mitochondria in response to complement-induced stress. In support, analysis of cytoplasmic mortalin by immunoblotting shows enhanced level of mortalin in the cytoplasm in complement-treated cells. Our data demonstrates that cells can sense complement activation at the plasma membrane and in response, swiftly send mortalin to this region in order to deactivate it.

Astrocytes from old Alzheimer's disease mice are impaired in Aß uptake and in neuroprotection.

In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aß42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aß clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aß uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.

Regulation of Complement-Dependent Cytotoxicity by MicroRNAs miR-200b, miR-200c, and miR-217.

The impact of microRNAs (miRNAs) known to regulate numerous biologic processes on complement-dependent cytotoxicity (CDC) was investigated in K562 cells. The C5b-9 complex is the executioner of CDC. Cells protect themselves from CDC by C5b-9 elimination, a process involving the mitochondrial chaperone mortalin/GRP75. Potential miR-200 (b and c) and miR-217 regulatory sites were identified in mortalin mRNA. Overexpression of miR-200b/c or miR-217 lowered the expression of mortalin mRNA. miRNA inhibitors for miR-200b, miR-200c, or miR-217 enhanced mortalin mRNA level. Unexpectedly, these miRNA modulators had no significant effect on mortalin protein level. Metabolic labeling analysis demonstrated that, to compensate for reduction in mortalin mRNA level, the cells increased the rate of synthesis of mortalin protein. Cells overexpressing miR-200b/c or miR-217 showed reduced sensitivity to CDC, whereas inhibition of miR-200c and miR-217 enhanced cell death. miR-200b/c overexpression reduced C5b-9 binding and enhanced its release from the cells and promoted mortalin relocation to the plasma membrane. Inhibition of miR-200 (b and c) and miR-217 had no effect on the expression level of the membrane complement-regulatory proteins CD46, CD55, and CD59. However, overexpression of miR-200b/c or miR-217 enhanced expression of CD46 and CD55 (not of CD59). Overall, the data demonstrate miRNA regulation of cell sensitivity to CDC. We identified miR-200b, miR-200c, and miR-217 as regulators of mortalin and, perhaps indirectly, of CD46 and CD55. Cell exposure to a sublytic dose of complement was shown to increase expression of miR-200 (b and c), suggesting that complement C5b-9 exerts a feedback-regulatory effect on these miRNAs.

Complement nomenclature 2014.

The first update since 1981 of the nomenclature used in the field of complement has been completed by the Complement Nomenclature Committee established under the auspices of the International Complement Society (ICS) and by the boards of the ICS and the European Complement Network (ECN). Recommended names of complement pathways, proteins, protein complexes, protein fragments and receptors are listed. Authors are urged to use these names in their published and presented works.

Mortalin/GRP75 binds to complement C9 and plays a role in resistance to complement-dependent cytotoxicity.

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.

Quantification of complement C5b-9 binding to cells by flow cytometry.

Interaction of the complement system, directly or indirectly (e.g., via antibodies), with cells activates the early and late complement components and culminates in the deposition of a membrane-spanning C5b-9 complex on the cell surface. At a high copy number, this C5b-9 will activate cell death, whereas at a low copy number, it will transmit various signals into cells. Quantification of C5b-9 deposition is useful for assessments of the capacity of cells and antibodies to activate complement. By using an antibody that identifies a novel antigen of the C5b-9 complex, the amount of C5b-9 complexes on cells can be quantified by flow cytometry. The detailed protocol is described in this chapter.

Construction and characterization of recombinant human C9 or C7 linked to single chain Fv directed to CD25.

Complement-dependent cytotoxicity (CDC) is a potent promoter of tumor clearance during monoclonal antibody therapy. Complement activation on antibody-bearing tumor cells results in formation of the membrane attack complex (MAC), which activates cell death. The complement activation cascade that bridges between antibody binding and MAC formation is regulated by complement inhibitors that are over-expressed on tumor cells. In order to bypass those complement regulators, we have designed an immunoconjugate composed of a humanized single chain Fv of an anti-Tac (CD25) monoclonal antibody fused at its C terminus either to complement protein C9 (scFv-C9) or to complement C7 (scFv-C7) and tagged with six histidines at the C terminal end. Recombinant scFv-C9 and scFv-C7 were expressed in 293T cells and purified. Both are shown to efficiently bind to CD25-positive tumor cells. In addition, scFv-C9, but not scFv-C7, increases MAC deposition on the cells and enhances complement-mediated cell death of target CD25-positive cells. Thus, scFv-C9 fusion protein is potentially a novel reagent for application in cancer immunotherapy.

Elevated levels of mitochondrial mortalin and cytosolic HSP70 in blood as risk factors in patients with colorectal cancer.

Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70 (HSP70). It protects cells from senescence and apoptosis and is overexpressed in cancer cells. Cell resistance to complement-dependent cytotoxicity depends on mortalin and during complement attack mortalin is released from cells. Our goal was to determine whether cancer patients have circulating mortalin in blood. The significance of mortalin in blood to survival prospects of colorectal cancer patients was evaluated. Occurrence of extracellular soluble HSP70 (sHSP70) is documented. We developed a sensitive ELISA for mortalin. The association between mortalin level and survival was subjected to the Cox proportional hazards analysis (univariate and multivariate analyses). Mortalin concentration in serum of colorectal cancer patients was 10-214 ng/ml. Survival data of the patients were known from an earlier study of sHSP70 in these samples. Cox regression analysis indicated that high mortalin (>60 ng/ml) is a risk factor for shorter survival. Serum levels of sHSP70 and mortalin in patients were independent variables. Concurrence of high sHSP70 and mortalin was associated with rapid disease progression (HR = 4, 2.04-8.45, p < 0.001). Addition of high sHSP70 and mortalin to a baseline model of age, sex and TNM stage, significantly (p < 0.001) enhanced the risk score to 8 (3.26-20.46). This is the first demonstration of circulating mortalin in cancer patients. Analysis of mortalin in blood, and even more so of mortalin and sHSP70, adds a high prognostic value to the TNM stage and will identify colorectal cancer patients at high risk of poor survival.

A role for the NF-κB pathway in cell protection from complement-dependent cytotoxicity.

Nucleated cells are equipped with several mechanisms that support their resistance to complement-dependent cytotoxicity (CDC). The role of the NF-κB pathway in cell protection from CDC was examined. Elevated sensitivity to CDC was demonstrated in cells lacking the p65 subunit of NF-κB or the IκB kinases IKKα or IKKß, and in cells treated with p65 small interfering RNA. Pretreatment with the IKK inhibitor PS-1145 also enhanced CDC of wild-type cells (WT) but not of p65(-/-) cells. Furthermore, reconstitution of p65 into p65(-/-) cells and overexpression of p65 in WT cells lowered their sensitivity to CDC. The postulated effect of p65 on the JNK-mediated death-signaling pathway activated by complement was examined. p65 small interfering RNA enhanced CDC in WT cells but not in cells lacking JNK. JNK phosphorylation induced by complement was more pronounced in p65(-/-) cells than in WT cells. The results indicate that the NF-κB pathway mediates cell resistance to CDC, possibly by suppressing JNK-dependent programmed necrotic cell death.

Caveolin-1 and dynamin-2 are essential for removal of the complement C5b-9 complex via endocytosis.

The complement system, an important element of both innate and adaptive immunity, is executing complement-dependent cytotoxicity (CDC) with its C5b-9 protein complex that is assembled on cell surfaces and transmits to the cell death signals. In turn, cells, and in particular cancer cells, protect themselves from CDC in various ways. Thus, cells actively remove the C5b-9 complexes from their plasma membrane by endocytosis. Inhibition of clathrin by transfection with shRNA or of EPS-15 with a dominant negative plasmid had no effect on C5b-9 endocytosis and on cell death. In contrast, inhibition of caveolin-1 (Cav-1) by transfection with an shRNA or a dominant negative plasmid sensitized cells to CDC and inhibited C5b-9 endocytosis. Similarly, both inhibition of dynamin-2 by transfection with a dominant negative plasmid or by treatment with Dynasore reduced C5b-9 endocytosis and enhanced CDC. C5b-9 endocytosis was also disrupted by pretreatment of the cells with methyl-ß-cyclodextrin or Filipin III, hence implicating membrane cholesterol in the process. Analyses by confocal microscopy demonstrated co-localization of Cav-1-EGFP with C5b-9 at the plasma membrane, in early endosomes, at the endocytic recycling compartment and in secreted vesicles. Further investigation of the process of C5b-9 removal by exo-vesiculation demonstrated that inhibition of Cav-1 and cholesterol depletion abrogated C5b-9 exo-vesiculation, whereas, over-expression of Cav-1 increased C5b-9 exo-vesiculation. Our results show that Cav-1 and dynamin-2 (but not clathrin) support cell resistance to CDC, probably by facilitating purging of the C5b-9 complexes by endocytosis and exo-vesiculation.