Landmarks of endosomal remodeling in the early phase of cytomegalovirus infection.

Cytomegaloviruses (CMVs) extensively rearrange the cellular membrane system to develop assembly compartment (AC), but the earliest events in this process are poorly characterized. Here, we demonstrate that murine CMV (MCMV) infection restrains endosomal trafficking of cargo molecules that travel along the recycling (TfR and MHC-I) and the late endosomal (EGFR, M6PR, Lamp1) circuit. Internalized cargo accumulates in Arf6-, Rab5-, Rab22A-, and Rab11-positive and Rab35-, Rab8-, and Rab10-negative juxtanuclear endosomes, suggesting the disruption of Arf/Rab regulatory cascade at the stage of sorting endosomes and the endosomal recycling compartment. Rearrangement of the endosomal system is initiated by an MCMV-encoded function very early in the infection. Our study, thus, establishes a set of landmarks of endosomal remodeling in the early phase of MCMV-infection which coincide with the Golgi rearrangement, suggesting that these perturbations are the earliest membrane reorganizations that may represent an initial step in the biogenesis of the AC.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

Late Endosomal Recycling of Open MHC-I Conformers.

With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd ) molecules, an open form of murine MHC-I allele, in fibroblast-like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC-I conformers. Kinetic modeling, using in-house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre-degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7-silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. J. Cell. Physiol. 232: 872-887, 2017. © 2016 Wiley Periodicals, Inc.

Cytomegalovirus immune evasion by perturbation of endosomal trafficking.

Cytomegaloviruses (CMVs), members of the herpesvirus family, have evolved a variety of mechanisms to evade the immune response to survive in infected hosts and to establish latent infection. They effectively hide infected cells from the effector mechanisms of adaptive immunity by eliminating cellular proteins (major histocompatibility Class I and Class II molecules) from the cell surface that display viral antigens to CD8 and CD4 T lymphocytes. CMVs also successfully escape recognition and elimination of infected cells by natural killer (NK) cells, effector cells of innate immunity, either by mimicking NK cell inhibitory ligands or by downregulating NK cell-activating ligands. To accomplish these immunoevasion functions, CMVs encode several proteins that function in the biosynthetic pathway by inhibiting the assembly and trafficking of cellular proteins that participate in immune recognition and thereby, block their appearance at the cell surface. However, elimination of these proteins from the cell surface can also be achieved by perturbation of their endosomal route and subsequent relocation from the cell surface into intracellular compartments. Namely, the physiological route of every cellular protein, including immune recognition molecules, is characterized by specific features that determine its residence time at the cell surface. In this review, we summarize the current understanding of endocytic trafficking of immune recognition molecules and perturbations of the endosomal system during infection with CMVs and other members of the herpesvirus family that contribute to their immune evasion mechanisms.

Luteolin ameliorates cisplatin-induced nephrotoxicity in mice through inhibition of platinum accumulation, inflammation and apoptosis in the kidney.

The aim of this study was to investigate the effects of flavone luteolin against cisplatin (CP)-induced kidney injury in mice. Luteolin at doses of 10mg/kg was administered intraperitoneally (ip) once daily for 3 days following single CP (10 or 20mg/kg) ip injection. Mice were sacrificed 24h after the last dose of luteolin. The CP treatment significantly increased serum creatinine and blood urea nitrogen and induced pathohistological changes in the kidneys. Renal oxidative/nitrosative stress was evidenced by decreased glutathione (GSH) levels and increased 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-HNE) formation as well as cytochrome P450 2E1 (CYP2E1) expression. The CP administration triggered inflammatory response in mice kidneys through activation of nuclear factor-kappaB (NF-κB) and overexpression of tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2). Simultaneously, the increase in renal p53 and caspase-3 expression indicated apoptosis of tubular cells. The administration of luteolin significantly reduced histological and biochemical changes induced by CP, decreased platinum (Pt) levels and suppressed oxidative/nitrosative stress, inflammation and apoptosis in the kidneys. These results suggest that luteolin is an effective nephroprotective agent, with potential to reduce Pt accumulation in the kidneys and ameliorate CP-induced nephrotoxicity.

Granulysin expression and the interplay of granulysin and perforin at the maternal-fetal interface.

Granulysin (GNLY) is a cytolytic/apoptotic molecule highly expressed in immune cells, particularly NK cells, at the maternal-fetal interface. The primary function of GNLY is to carry out lysis or apoptosis induction in target cells, tumor cells or cells infected by intracellular pathogens. To exert some of its functions GNLY needs to collaborate with perforin. The purpose of this study was to determine: (a) the expression of GNLY at the gene and protein levels at the maternal-fetal interface, (b) the relationship(s) between GNLY and perforin, and (c) GNLY secretion by NK cells stimulated by the NK-sensitive K562 cell line and its HLA-C and HLA-G transfectants. GNLY and perforin genes were found to be highly activated at the interface. GNLY mRNA was present at significantly higher levels compared with other cytolytic/apoptotic molecules. Confocal microscopy analysis showed that most first trimester pregnancy decidual lymphocytes simultaneously contained both GNLY and perforin protein in their cytoplasm, with a punctuate pattern consistent with granule localization. In contrast to peripheral blood, in unstimulated decidual lymphocytes GNLY and perforin rarely co-localized (10% of GNLY-positive cells and 20% of perforin-positive cells were positive for both proteins). Contact between decidual lymphocytes and K562 cells caused GNLY and perforin to be expressed in the same granules (approximately 50% co-localization), i.e., to attain the pattern seen in peripheral blood lymphocytes. The abundant GNLY secretion by decidual NK cells compared with peripheral blood NK cells after 2h of contact with the NK-sensitive K562 cells and K562 transfectants was striking.

Endosomal trafficking of open Major Histocompatibility Class I conformers--implications for presentation of endocytosed antigens.

Major Histocompatibility Class I (MHC-I) molecules are present at the cell surface either as fully conformed trimolecular complexes composed of heavy chain, beta-2-microglobulin (ß2m) and antigenic peptide or as various open forms, devoid of the peptide and/or ß2m. While the role of fully conformed MHC-I is well studied, the physiological role of open conformers is neglected. We have shown that fully conformed MHC-I and open MHC-I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC-I from the early endosomal/juxtanuclear recycling route. As a result, open MHC-I conformers are internalized with a higher rate than fully conformed counterparts. Although the majority of internalized open MHC-I is directed into the acidic late endosomal (LE) compartments, only a fraction of them is degraded. Namely, a significant fraction of open MHC-I is present in a subset of LEs with the capacity of recycling and/or exocytosis. Therefore, it should be examined whether exogenous peptide loading may occur during traveling of MHC-I proteins through LE compartments, especially in a subset of less acidic LEs that detach from the core of perinuclear acidic LEs and migrate toward the cell periphery. Given that the acidic LE environment is not favorable for peptide loading, an endosomal compartment with the recycling capacity and less acidic environment that allows stabilization of newly formed trimolecular complexes is proper site for exogenous peptide loading. We propose that a LE compartment which collect and retain open MHC-I conformers should be taken into consideration as a site of exogenous peptide loading.

Expression of cytolytic protein-perforin in peripheral blood lymphocytes in severe traumatic brain injured patients.

PURPOSE: The purpose of this study was to investigate the changes of cytotoxic protein-perforin in peripheral blood lymphocytes in severe TBI patients and possible correlation between severity of TBI and perforin expression. METHODS: Flow cytometry was used for simultaneous detection of intracellular perforin and cell surface antigens of peripheral blood lymphocytes of 20 severe TBI patients on day 1, 4 and 7 after the onset of injury. Peripheral blood mononuclear cells from 20 healthy volunteers were used as control. Clinical and laboratory parameters were also recorded. RESULTS: There was a statistically significant decrease of perforin-positive lymphocytes including T, natural killer (NK) and NKT cells on day 4 as compared with day 1 after the brain injury or healthy controls. On day 7, perforin expression was restored in lymphocyte of cytotoxic phenotype (CD8(+) T lymphocytes, NK cells, and NKT cells) compared with day 1. High positive correlation was found between the severity of TBI and frequency of perforin-positive cells on day 4 when the occurrence of the intra-hospital infections was the highest. CONCLUSION: Severe TBI significantly decreases perforin expression in T lymphocytes, NK and NKT cells, which indicate a possible mechanism underlying the high susceptibility to infections.

Early endosomal rerouting of major histocompatibility class I conformers.

Major histocompatibility class I (MHC-I) molecules are present at the cell surface both as fully conformed trimolecular complexes composed of heavy chain (HC), beta-2-microglobulin (ß2m) and peptide, and various open forms, devoid of peptide and/or ß2m (open MHC-I conformers). Fully conformed MHC-I complexes and open MHC-I conformers can be distinguished by well characterized monoclonal antibody reagents that recognize their conformational difference in the extracellular domain. In the present study, we used these tools in order to test whether conformational difference in the extracellular domain determines endocytic and endosomal route of plasma membrane (PM) proteins. We analyzed PM localization, internalization, endosomal trafficking, and recycling of human and murine MHC-I proteins on various cell lines. We have shown that fully conformed MHC-I and open MHC-I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC-I conformers from the recycling route. This segregation is associated with their partitioning into the membranes of different compositions. As a result, the open MHC-I conformers internalized with higher rate than fully conformed counterparts. Thus, our data suggest the existence of conformation-based protein sorting mechanism in the endosomal system.

Segregation of open Major Histocompatibility Class I conformers at the plasma membrane and during endosomal trafficking reveals conformation-based sorting in the endosomal system.

Fully conformed Major Histocompatibility Class I molecules are complexes of heavy chain non-covalently associated with the peptide and beta-2-microglobulin. Conformational change in the extracellular domain of heavy chain leads to their disassembly and formation of open conformers, a process that physiologically occurs in normal cells and results in their presence at the cell surface. In this study we characterized endosomal trafficking of open conformers of a murine class I allele in order to examine whether conformational change in the extracellular domain of a membrane glycoprotein determines its endosomal sorting. Open conformers segregated from their fully conformed counterparts at the plasma membrane and in endosomes by sequestration in lipid-organized membrane environment. Consequently, open conformers constitutively internalized via distinct clathrin-independent endocytic carriers and converged into "classical" early endosomes together with transferrin receptor and cholera-toxin B subunit. In early endosomes, open conformers were excluded from recycling and diverted towards late endosomes. Due to lack of recycling, open conformers were constitutively internalized at a higher rate than full conformed proteins. Concanamycin A, methyl-ß-cyclodextrin and sphingomyelinase treatment prevented segregation of open conformers in vacuolar early endosomes indicating that acidic endosomal environment and membrane composition are critical for the maintenance of the sorting mechanism. In the absence of endosomal acidification open conformers partitioned into lipid disordered membrane composition of early endosomes. Thus, our data suggest for the existence of a lipid-dependent mechanism in the endosomal system that distinguish membrane proteins based on conformation of their extracellular domain.

Murine cytomegalovirus perturbs endosomal trafficking of major histocompatibility complex class I molecules in the early phase of infection.

Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.

Constitutive internalization of murine MHC class I molecules.

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (K(d), D(d), full L(d), and empty L(d)) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. K(d) and D(d) molecules are more stable at the cell surface than L(d) molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (K(d), D(d), and L(d)) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty L(d) molecules was inhibited by filipin, indicating that non-conformed MHC class I molecules require intact cholesterol-rich membrane microdomains for their constitutive internalization. Thus, conformed and non-conformed MHC class I molecules use different endocytic pathways for constitutive internalization.

Inhibition of protein kinases C prevents murine cytomegalovirus replication.

For successful establishment of infection and initiation of the replication cycle, murine cytomegalovirus (MCMV) utilizes cellular structures and functions, including cell-membrane penetration, capsid dismantling and cytosolic transport of viral DNA into the nucleus. These early events of MCMV infections are dependent on cellular regulatory mechanisms, primarily protein phosphorylation. In the present study, protein kinase inhibitors were used to explore the role of protein phosphorylation mediated by protein kinases C (PKCs) in the very early events of MCMV infection. Inhibitory effects were determined by immunofluorescence and Western blot analysis of MCMV IE1 and E1 protein expression and by production of infectious virions in cell culture. It was found that H-7, a broadly specific inhibitor of cellular protein kinases, prevented virus replication in a dose-dependent and reversible manner, and that the block in replication occurred very early in infection. More specific PKC inhibitors (sangivamycin, calphostin C and bisindolylmaleimide II), Ca(2+)/calmodulin inhibitors (EDTA and W7) and phorbol esters (PMA) were used to dissect PKC-subclass contribution in the very early events of MCMV replication. The results indicate that the role of diacylglycerol/phorbol ester-dependent but calcium-independent PKCs is essential for establishment of MCMV infection in the host cell, starting at a very early stage of infection.

Distinct pathways for constitutive endocytosis of fully conformed and non-conformed L(d) molecules.

PROBLEM: To characterize the constitutive internalization of major histocompatibility complex (MHC) class I molecules, we have studied the expression of completely conformed (full) and unconformed (empty) L(d) molecules on non-polarized murine P815 cells. METHODS OF STUDY: Spontaneous endocytosis of L(d) molecules was induced by cycloheximide, an inhibitor of protein synthesis, and their disappearance from the cell surface was determined by flow cytometry. In order to investigate the mechanism of internalization, a palette of inhibitors of endocytosis and vesicular transport was used. RESULTS: Inhibitors of clathrine endocytosis did not influence the internalization of L(d) molecules. Inhibitors of caveolar endocytosis and inhibitors of endolysosomal degradation prevented down-regulation of empty, but not of full L(d) molecules. CONCLUSIONS: Empty L(d) molecules are internalized mostly by caveolar endocytosis and full L(d) molecules use a different pathway, neither clathrine-mediated nor caveolar. After internalization, full L(d) molecules are probably degraded and empty L(d) molecules recycle between endosomal compartment and the cell surface before they enter into the degradation compartment.