Extracellular Vesicles Isolated from Equine Adipose-Derived Stromal Stem Cells (ASCs) Mitigate Tunicamycin-Induced ER Stress in Equine Corneal Stromal Stem Cells (CSSCs).

Corneal ulcers, characterized by severe inflammation of the cornea, can lead to serious, debilitating complications and may be vision-threatening for horses. In this study, we aimed to investigate the role of endoplasmic reticulum (ER) stress in corneal stem progenitor cell (CSSC) dysfunction and explore the potential of equine adipose-derived stromal stem cell (ASC)-derived extracellular vesicles (EVs) to improve corneal wound healing. We showed that CSSCs expressed high levels of CD44, CD45, and CD90 surface markers, indicating their stemness. Supplementation of the ER-stress-inducer tunicamycin to CSSCs resulted in reduced proliferative and migratory potential, accumulation of endoplasmic reticulum (ER)-stressed cells in the G0/G1 phase of the cell cycle, increased expression of proinflammatory genes, induced oxidative stress and sustained ER stress, and unfolded protein response (UPR). Importantly, treatment with EVs increased the proliferative activity and number of cells in the G2/Mitosis phase, enhanced migratory ability, suppressed the overexpression of proinflammatory cytokines, and upregulated the anti-inflammatory miRNA-146a-5p, compared to control and/or ER-stressed cells. Additionally, EVs lowered the expression of ER-stress master regulators and effectors (PERK, IRE1, ATF6, and XBP1), increased the number of mitochondria, and reduced the expression of Fis-1 and Parkin, thereby promoting metabolic homeostasis and protecting against apoptosis in equine CSSCs. Our findings demonstrate that MSCs-derived EVs represent an innovative and promising therapeutic strategy for the transfer of bioactive mediators which regulate various cellular and molecular signaling pathways.

The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi.

Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.

A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1.

The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication.IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.

Promiscuity of the catalytic Sec7 domain within the guanine nucleotide exchange factor GBF1 in ARF activation, Golgi homeostasis, and effector recruitment.

The integrity of the Golgi and trans-Golgi network (TGN) is disrupted by brefeldin A (BFA), which inhibits the Golgi-localized BFA-sensitive factor (GBF1) and brefeldin A-inhibited guanine nucleotide-exchange factors (BIG1 and BIG2). Using a cellular replacement assay to assess GBF1 functionality without interference from the BIGs, we show that GBF1 alone maintains Golgi architecture; facilitates secretion; activates ADP-ribosylation factor (ARF)1, 3, 4, and 5; and recruits ARF effectors to Golgi membranes. Unexpectedly, GBF1 also supports TGN integrity and recruits numerous TGN-localized ARF effectors. The impact of the catalytic Sec7 domain (Sec7d) on GBF1 functionality was assessed by swapping it with the Sec7d from ARF nucleotide-binding site opener (ARNO)/cytohesin-2, a plasma membrane GEF reported to activate all ARFs. The resulting chimera (GBF1-ARNO-GBF1 [GARG]) targets like GBF1, supports Golgi/TGN architecture, and facilitates secretion. However, unlike GBF1, GARG activates all ARFs (including ARF6) at the Golgi/TGN and recruits additional ARF effectors to the Golgi/TGN. Our results have general implications: 1) GEF's targeting is independent of Sec7d, but Sec7d influence the GEF substrate specificity and downstream effector events; 2) all ARFs have access to all membranes, but are restricted in their distribution by the localization of their activating GEFs; and 3) effector association with membranes requires the coincidental presence of activated ARFs and specific membrane identifiers.

Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity.

Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based "replacement" assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1.

The ARF guanine nucleotide exchange factor GBF1 is targeted to Golgi membranes through a PIP-binding domain.

ADP-ribosylation factors (ARF) GTPases are activated by guanine nucleotide exchange factors (GEFs) to support cellular homeostasis. Key to understanding spatio-temporal regulation of ARF signaling is the mechanism of GEF recruitment to membranes. Small GEFs are recruited through phosphoinositide (PIP) binding by a pleckstrin homology (PH) domain downstream from the catalytic Sec7 domain (Sec7d). The large GEFs lack PH domains, and their recruitment mechanisms are poorly understood. We probed Golgi recruitment of GBF1, a GEF catalyzing ARF activation required for Golgi homeostasis. We show that the homology downstream of Sec7d-1 (HDS1) regulates Golgi recruitment of GBF1. We document that GBF1 binds phosphoinositides, preferentially PI3P, PI4P and PI(4,5)P2, and that lipid binding requires the HDS1 domain. Mutations within HDS1 that reduce GBF1 binding to specific PIPs in vitro inhibit GBF1 targeting to Golgi membranes in cells. Our data imply that HDS1 and PH domains are functionally analogous in that each uses lipid-based membrane information to regulate GEF recruitment. Lipid-based recruitment of GBF1 extends the paradigm of lipid regulation to small and large GEFs and suggests that lipid-based mechanisms evolved early during GEF diversification. This article has an associated First Person interview with the first author of the paper.

αII-spectrin in T cells is involved in the regulation of cell-cell contact leading to immunological synapse formation?

T-lymphocyte activation after antigen presentation to the T-Cell Receptor (TCR) is a critical step in the development of proper immune responses to infection and inflammation. This dynamic process involves reorganization of the actin cytoskeleton and signaling molecules at the cell membrane, leading to the formation of the Immunological Synapse (IS). The mechanisms regulating the formation of the IS are not completely understood. Nonerythroid spectrin is a membrane skeletal protein involved in the regulation of many cellular processes, including cell adhesion, signaling and actin cytoskeleton remodeling. However, the role of spectrin in IS formation has not been explored. We used molecular, imaging and cellular approaches to show that nonerythroid αII-spectrin redistributes to the IS during T-cell activation. The redistribution of spectrin coincides with the relocation of CD45 and LFA-1, two components essential for IS formation and stability. We assessed the role of spectrin by shRNA-mediated depletion from Jurkat T cells and show that spectrin-depleted cells exhibit decreased adhesion and are defective in forming lamellipodia and filopodia. Importantly, IS formation is impaired in spectrin-depleted cells. Thus, spectrin may be engaged in regulation of distinct events necessary for the establishment and maturity of the IS: besides the involvement of spectrin in the control of CD45 and LFA-1 surface display, spectrin acts in the establishment of cell-cell contact and adhesion processes during the formation of the IS.

[Liposomes as non-viral carriers for genetic drugs]. / Liposomy jako niewirusowe systemy dostarczania leków genetycznych.

Methods in cancer therapy particularly in recent years, are rapidly changing, due to the need of design of new, more effective therapeutic strategies. Very promising approach to treatment of the neoplastic diseases is antisense gene therapy. Due to the low toxicity of treatment and eliminating not only the symptoms but also the molecular causes of the disease it may represent a breakthrough in cancer therapies. Delivery of a therapeutic DNA or RNA oligonucleotides to the target cells in vivo requires suitable carrier system. Non-viral drug carriers are increasingly used in new systems of targeted gene therapy. This review presents new generation of non-viral carriers, and is focused on immunoliposomes finding potential application in targeted gene therapy.

Novel antisense therapeutics delivery systems: In vitro and in vivo studies of liposomes targeted with anti-CD20 antibody.

Antisense gene therapy using molecules such as antisense oligodeoxynucleotides, siRNA or miRNA is a very promising strategy for the treatment of neoplastic diseases. It can be combined with other treatment strategies to enhance therapeutic effect. In acute leukemias, overexpression of the antiapoptotic gene BCL2 is observed in more than 70% of cases. Therefore, reduction of the Bcl-2 protein level could, in itself, prevent the development of cancer or could possibly help sensitize cancer cells to apoptosis inducers. The main objective of our work is to develop therapeutic liposome formulations characterized by high transfection efficiency, stability in the presence of serum, as well as specificity and toxicity for target (leukemic) cells. Each of our liposomal formulations consists of a core composed of antisense oligonucleotides complexed by either cationic lipid, DOTAP, or a synthetic polycation, polyethyleneimine, encapsulated within liposomes modified with polyethylenoglycol. In addition, the liposomal shells are enriched with covalently-bound antibodies recognizing a well characterized bio-marker, CD20, exposed on the surface of leukemia cells. The resulting immunoliposomes selectively and effectively reduced the expression of BCL2 in target cells. Model animal experiments carried out on mice-engrafted tumors expressing the specific marker showed high efficiency of the liposome formulations against specific tumor development. In conclusion, we show that lipid formulations based on a polyplex or lipoplex backbone additionally equipped with antibodies are promising non-viral vectors for specific oligonucleotide transfer into human tumor cells.