Study on the role of protein deiminases in the nervous system

"Study on the role of protein deiminases in the nervous system" Citrullination (deimination) is the conversion of protein-bound arginine to citrulline. Citrullination is catalyzed by a family of calcium-dependent enzymes, the peptidylarginine deiminases (PADs). In mammals there are five PADs (PAD1, PAD2, PAD3, PAD4 and PAD6), whereas only 3 are found in chicken (PAD1-3). PAD isoenzymes are widely distributed in mammalian tissues and several studies suggest that citrullination occurs in extreme conditions such as during apoptosis, and during differentiation, when there is an increase in intracellular calcium concentration. Citrullination of different PAD target proteins has been associated with certain diseases, such as Alzheimer's disease, multiple sclerosis, rheumatoid arthritis and psorias. In humans, citrullination might be an early marker in neurodegenerative diseases. The role of PADs and citrullination in some human diseases is poorly understood and the physiological roles of PADs have yet to be fully investigated. At present citrullination is believed to play a role in myelin sheath formation and during keratinocytes terminal differentiation. Recent studies in mouse have suggested that PAD4 regulates histone methylation at the p21/WAF1/CIP1 promoters in a p53-dependent manner. The group of PF at UCL Institute of Child Health in London has identified PAD3 among calcium-dependent molecules differentially regulated in response to spinal cord injury at stages of development permissive (E11) and non-permissive (E15) for regeneration in chick embryos. Consistent with the up-regulation of PAD3 in spinal cords injured at E15, more extensive citrullination was observed after injury at this stage. This paralleled high apoptosis and significant tissues loss in injured E15 spinal cords. Following treatment at the time of injury with a PAD inhibitor, Cl-amidine, the secondary injury response in E15 spinal cord was greatly reduced. The aim of my project was first to see if it was possible to model the injury response in neural cells in vitro to eventually establish a model for studying PAD, citrullination and injury response in human neural cells and secondly I tried to better characterize expression pattern and possible role(s) of PAD, focusing mainly on PAD3, in neural cell death and survival initially using neural progenitor cells derived from E7 (embryonic day 7) chick midbrain (cMB cells), which were shown to express PAD3 both at the protein and mRNA level, and subsequently in a human neural tumor cell line, the neuroblastoma cell line LAN5. To this purposes we first studied the effects of PAD inhibition, using Cl-amidine, and PAD activation, using thapsigargin to raise intracellular calcium, on expression and cellular localization of PAD and on citrullination of a PAD target, histone 3 (H3) in cMB cells. In some experiments chick midbrain cells from E7 embryos were treated also with the p53 inhibitor, pifithrin, to assess whether p53 is required for PAD activity. Thapsigargin treatment induced significant cell death, but this was reduced by pretreating the cells with Cl-amidine. Reduction of cell death upon PAD inhibition parallels the in vivo finding in the injured chick spinal cord. Significantly, PAD3 mainly localized to the nucleus in cMB cells at passage 12 following thapsigargin treatment, whereas it remained largely cytoplasmatic in controls and cells pre-treated with Cl-amidine. Citrullinated H3 (CitH3) was not restricted to the nucleus in thapsigargin-treated cells while it is definetly nuclear in control and CL-amidine treated cells. These results suggest that the PAD-associated apoptotic effect is likely due to PAD activity in the nucleus and that increased citrullination of H3 results in its export from the nucleus. As cMB cells were found to become senescent and die around passage 30 (P30), we investigated whether expression of PAD3 and CitH3 changed with time in culture. At P30 PAD3 was largely nuclear both in control and treated cells (thapsigargin/Cl-amidine), and CitH3 distribution resembeld that observed at P12 following thapsigargin-treatment. No changes in PAD3 localization were observed upon thapsigargin/Cl-amidine-treatments at P30, unlike at P12 where PAD3 localized in the nucleus only in thapsigargine-treated cells. Western blotting showed no differences in the amount of CitH3 among the different treatments. These results suggest an increased nuclear activity of PAD3 and increased turnover of CitH3 with aging that may be associated with cellular senescence and death. As certain tumor cell lines were reported to express PADs, we assessed expression of PAD3 and PAD4, another PAD that can localize to the nucleus, and the response to thapsigargin and Cl-amidine treatment in LAN5 cells. PAD3 and PAD4 appeared to be mainly perinuclear, and their expression and localization was not affected by any drug combination (assessed by immunocytochemistry). Western blotting and immunocytochemistry showed that also CitH3 amount and localization were not affected in treated cells. In addition to an effect on cell death, PAD activity was found to play a role in cell adhesion. When plated in the presence of Cl-amidine, most cMB cells did not attach to the dish, but were able to do so upon removal of Cl-amidine. Therefore their adhesion ability rather than survival appear to be affected. When cells were grown in neural differentiation medium (serum-free) to study a possible role of PAD in differentiation, such recovery was not observed. Howewer, Given that PAD3 expression was observed both in cell positive and negative for the neuronal marker, ß3-tubulin, and that in the presence of serum Cl-amidine did not affect expression of ß3-tubulin, as indicated by immunocytochemistry and Western blotting, a role for PAD in neuronal differentiation does not seem to be likely. The expression pattern of PAD3 during chick embryonic development was assessed by mRNA analysis and RT-PCR in tissues dissected from embryos in variuos stage of development. Valentina Millart

The Golgi in Cell Migration: Regulation by Signal Transduction and Its Implications for Cancer Cell Metastasis

Migration and invasion are fundamental features of metastatic cancer cells. The Golgi apparatus, an organelle involved in posttranslational modification and sorting of proteins, is widely accepted to regulate directional cell migration. In addition, mounting evidence suggests that the Golgi is a hub for different signaling pathways. In this paper we will give an overview on how polarized secretion and microtubule nucleation at the Golgi regulate directional cell migration. We will review different signaling pathways that signal to and from the Golgi. Finally, we will discuss how these signaling pathways regulate the role of the Golgi in cell migration and invasion. We propose that by identifying regulators of the Golgi, we might be able to uncover unappreciated modulators of cell migration. Uncovering the regulatory network that orchestrates cell migration is of fundamental importance for the development of new therapeutic strategies against cancer cell metastasis

Rabaptin5 targets autophagy to damaged endosomes and Salmonella vacuoles via FIP200 and ATG16L1

Selective autophagy of damaged organelles is important to maintain cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here, we identify two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Autophagy of early endosomes damaged by chloroquine or monensin treatment requires Rabaptin5 and particularly a short sequence motif that binds to the WD domain of ATG16L1. Rabaptin5 and its interaction with ATG16L1 further contributes to the autophagic elimination of Salmonella enterica early after infection, when it resides in phagosomes with early endosomal characteristics. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective targeting of autophagy to damaged early endosomes and phagosomes

BPAG1a and b Associate with EB1 and EB3 and Modulate Vesicular Transport, Golgi Apparatus Structure, and Cell Migration in C2.7 Myoblasts

BPAG1a and BPAG1b (BPAG1a/b) constitute two major isoforms encoded by the dystonin (Dst) gene and show homology with MACF1a and MACF1b. These proteins are members of the plakin family, giant multi-modular proteins able to connect the intermediate filament, microtubule and microfilament cytoskeletal networks with each other and to distinct cell membrane sites. They also serve as scaffolds for signaling proteins that modulate cytoskeletal dynamics. To gain better insights into the functions of BPAG1a/b, we further characterized their C-terminal region important for their interaction with microtubules and assessed the role of these isoforms in the cytoskeletal organization of C2.7 myoblast cells. Our results show that alternative splicing does not only occur at the 5′ end of Dst and Macf1 pre-mRNAs, as previously reported, but also at their 3′ end, resulting in expression of additional four mRNA variants of BPAG1 and MACF1. These isoform-specific C-tails were able to bundle microtubules and bound to both EB1 and EB3, two microtubule plus end proteins. In the C2.7 cell line, knockdown of BPAG1a/b had no major effect on the organization of the microtubule and microfilament networks, but negatively affected endocytosis and maintenance of the Golgi apparatus structure, which became dispersed. Finally, knockdown of BPAG1a/b caused a specific decrease in the directness of cell migration, but did not impair initial cell adhesion. These data provide novel insights into the complexity of alternative splicing of Dst pre-mRNAs and into the role of BPAG1a/b in vesicular transport, Golgi apparatus structure as well as in migration in C2.7 myoblasts

RABEP1/Rabaptin5: a link between autophagy and early endosome homeostasis

Selective autophagy of damaged organelles assures maintenance of cellular homeostasis in eukaryotes. While the mechanisms by which cells selectively remove dysfunctional mitochondria, lysosomes, endoplasmic reticulum and other organelles has been well characterized, little is known about specific autophagy of damaged early endosomes. In our recent study, we uncovered a new role for RABEP1/Rabaptin5, a long-established regulator of early endosome function, in targeting the autophagy machinery to early endosomes damaged by chloroquine or by internalized; Salmonella; via interaction with RB1CC1/FIP200 and ATG16L1

Regulation of Sec16 levels and dynamics links proliferation and secretion

We currently lack a broader mechanistic understanding of the integration of the early secretory pathway with other homeostatic processes such as cell growth. Here, we explore the possibility that Sec16A, a major constituent of endoplasmic reticulum exit sites (ERES), acts as an integrator of growth factor signalling. Surprisingly, we find that Sec16A is a short-lived protein that is regulated by growth factors in a manner dependent on Egr family transcription factors. We hypothesize that Sec16A acts as a central node in a coherent feed-forward loop that detects persistent GF stimuli to increase ERES number. Consistent with this notion, Sec16A is also regulated by short-term growth factor treatment that leads to increased turnover of Sec16A at ERES. Finally, we demonstrate that Sec16A depletion reduces, while its overexpression increases proliferation. Together with our finding that growth factors regulate Sec16A levels and its dynamics on ERES, we propose this protein as an integrator linking growth factor signalling and secretion. This provides a mechanistic basis for the previously proposed link between secretion and proliferation

FIG4 is a hepatitis C virus particle-bound protein implicated in virion morphogenesis and infectivity with cholesteryl ester modulation potential

There is growing evidence that virus particles also contain host cell proteins, which provide viruses with certain properties required for entry and release. A proteomic analysis performed on double-gradient-purified hepatitis C virus (HCV) from two highly viraemic patients identified the phosphatidylinositol 3,5-bisphosphate 5-phosphatase FIG4 (KIAA0274) as part of the viral particles. We validated the association using immunoelectron microscopy, immunoprecipitation and neutralization assays in vitro as well as patient-derived virus particles. RNA interference-mediated reduction of FIG4 expression decreased cholesteryl ester (CE) levels along with intra- and extracellular viral infectivity without affecting HCV RNA levels. Likewise, overexpressing FIG4 increased intracellular CE levels as well as intra- and extracellular viral infectivity without affecting viral RNA levels. Triglyceride levels and lipid droplet (LD) parameters remained unaffected. The 3,5-bisphosphate 5-phosphatase active site of FIG4 was found to strongly condition these results. Whilst FIG4 was found to localize to areas corresponding to viral assembly sites, at the immediate vicinity of LDs in calnexin-positive and HCV core-positive regions, no implication of FIG4 in the secretory pathway of the hepatocytes could be found using either FIG4-null mice, in vitro morphometry or functional assays of the ERGIC/Golgi compartments. This indicates that FIG4-dependent modulation of HCV infectivity is unrelated to alterations in the functionality of the secretory pathway. As a result of the documented implication of CE in the composition and infectivity of HCV particles, these results suggest that FIG4 binds to HCV and modulates particle formation in a CE-related manner