Gordana Blagojević Zagorac

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.

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.

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 (β2m) and peptide, and various open forms, devoid of peptide and/or β2m (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. J. Cell. Physiol. 227: 2953–2964, 2012.

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 (β2m) and antigenic peptide or as various open forms, devoid of the peptide and/or β2m. 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.

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.

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.

Hepatic Expression of Metallothionein I/II, Glycoprotein 96, IL-6, and TGF-β in Rat Strains with Different Susceptibilities to Experimental Autoimmune Encephalomyelitis

In a search of peripheral factors that could be responsible for the discrepancy in susceptibility to EAE in Albino Oxford (AO) and Dark Agouti (DA) rats, we estimated the expression of metallothioneins I/II (MT), heat shock protein-gp96, interleukin (IL)-6, and transforming growth factor (TGF)-β in the livers of these animals. Rats were immunized with bovine brain homogenate (BBH) emulsified in complete Freund adjuvant (CFA) or only with CFA. Western blot and immunohistochemical analyses were done on day 12 after the immunization, as well as in intact rats. The data have shown that during the first attack of EAE only the EAE prone-DA rats markedly upregulated the hepatic MTs, gp96, IL-6, and TGF-β. In contrast, AO rats had a significantly higher expression of MT I/II, IL-6, and TGF-β in intact liver (P < 0,001), suggesting that the greater constitutive expression of these proteins contributed to the resistance of EAE. Besides, since previously we found that AO rats reacted on immunization by an early upregulation of TGF-β on several hepatic structures (vascular endothelium, Kupffer cells, and hepatocytes), the data suggest that the specific hepatic microenvironment might contribute also to the faster recovery of these rats from EAE.