Hiba El Hajj

The rhoptry neck protein RON4 relocalizes at the moving junction during Toxoplasma gondii invasion

Host cell invasion in the Apicomplexa is unique in its dependency on a parasite actin‐driven machinery and in the exclusion of most host cell membrane proteins during parasitophorous vacuole (PV) formation. This exclusion occurs at a junction between host cell and parasite plasma membranes that has been called the moving junction, a circumferential zone which forms at the apical tip of the parasite, moves backward and eventually pinches the PV from the host cell membrane. Despite having been described by electron microscopic studies 30 years ago, the molecular nature of this singular structure is still enigmatic. We have obtained a monoclonal antibody that recognizes the moving junction of invading tachyzoites of Toxoplasma gondii, in a pattern clearly distinct from those described so far for microneme and rhoptry proteins. The protein recognized by this antibody has been affinity purified. Mass spectrometry analysis showed that it is a rhoptry neck protein (RON4), a hypothetical protein with homologues restricted to Apicomplexa. Our findings reveals for the first time the participation of rhoptry neck proteins in moving junction formation and strongly suggest the conservation of this structure at the molecular level among Apicomplexa.

ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243–539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence.

Phase 2 study of the efficacy and safety of the combination of arsenic trioxide, interferon alpha, and zidovudine in newly diagnosed chronic adult T-cell leukemia/lymphoma (ATL)

Adult T-cell leukemia/lymphoma (ATL) is resistant to chemotherapy and carries a dismal prognosis particularly for the acute and lymphoma subtypes. Promising results were obtained with the combination of zidovudine and interferon-alpha. Chronic ATL has a relatively better outcome, but poor long-term survival is noted when patients are managed with a watchful-waiting policy or with chemotherapy. In ATL cell lines, arsenic trioxide shuts off constitutive NF-kappaB activation and potentiates interferon-alpha apoptotic effects through proteasomal degradation of Tax. Clinically, arsenic/interferon therapy exhibits some efficacy in refractory aggressive ATL patients. These results prompted us to investigate the efficacy and safety of the combination of arsenic, interferon-alpha, and zidovudine in 10 newly diagnosed chronic ATL patients. An impressive 100% response rate was observed including 7 complete remissions, 2 complete remissions but with more than 5% circulating atypical lymphocytes, and 1 partial response. Responses were rapid and no relapse was noted. Side effects were moderate and mostly hematologic. In conclusion, treatment of chronic ATL with arsenic, interferon-alpha, and zidovudine is feasible and exhibits an impressive response rate with moderate toxicity. Long-term follow up will clarify whether this will translate to disease cure. Overall, these clinical results strengthen the concept of oncogene-targeted cancer therapy.

Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane

Toxoplasma gondii, as many intracellular parasites, is separated from the cytosol of its host cell by a parasitophorous vacuole membrane (PVM). This vacuole forms during host cell invasion and parasite apical organelles named rhoptries discharge proteins that associate with its membrane during this process. We report here the characterization of the rhoptry protein ROP5, which is a new member of the ROP2 family. Contrasting with what is known for other ROP2 family proteins, ROP5 is not processed during trafficking to rhoptries. We show here that ROP5 is secreted during invasion and associates with the PVM. Using differential permeabilization of infected cells, we have shown that ROP5 exposes its C‐terminus towards the host cell cytoplasm, which corresponds to a reverse topology compared with ROP2 and ROP4. Taken together with recent modelling data suggesting that the C‐terminal hydrophobic domain hitherto described as transmembrane may correspond to a hydrophobic helix buried in the catalytic domain of kinase‐related proteins, these findings call for a reappraisal of the current view of ROP2 family proteins association with the PVM.

Therapy-induced selective loss of leukemia-initiating activity in murine adult T cell leukemia

Treatment with a combination of interferon-α and arsenic trioxide ablates leukemia-initiating activity before reducing primary tumor bulk in a murine model of adult T cell leukemia.

Molecular Signals in the Trafficking of Toxoplasma gondii Protein MIC3 to the Micronemes

ABSTRACT The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.

Tax ubiquitylation and SUMOylation control the dynamic shuttling of Tax and NEMO between Ubc9 nuclear bodies and the centrosome

The HTLV-I oncoprotein Tax is critical for T cell transformation, acting mainly through NEMO binding and subsequent NFB activation. Tax localizes to Tax nuclear bodies and to the centrosome and is subjected to ubiquitylation and SUMOylation that are both necessary for complete transcriptional activation. By using the photoconvertible fluorophore Dendra-2 coupled with live video confocal microscopy, we show for the first time that the same Tax molecule shuttles among Tax nuclear bodies and between these nuclear bodies and the centrosome depending on its post-translational modifications. Ubiquitylation targets Tax to nuclear bodies to which NEMO is recruited and subsequently SUMOylated. We also demonstrate that Tax nuclear bodies contain the SUMOylation machinery including SUMO and the SUMO conjugating enzyme Ubc9, strongly suggesting that these nuclear bodies represent sites of active SUMOylation. Finally, both ubiquitylation and SUMOylation of Tax control NEMO targeting to the centrosome. Altogether, we are proposing a model where both ubiquitylation and SUMOylation of Tax control the shuttling of Tax and NEMO between the cytoplasmic and nuclear compartments.

Controversies in Targeted Therapy of Adult T Cell Leukemia/Lymphoma: ON Target or OFF Target Effects?

Adult T cell leukemia/lymphoma (ATL) represents an ideal model for targeted therapy because of intrinsic chemo-resistance of ATL cells and the presence of two well identified targets: the HTLV-I retrovirus and the viral oncoprotein Tax. The combination of zidovudine (AZT) and interferon-alpha (IFN) has a dramatic impact on survival of ATL patients. Although the mechanism of action remains unclear, arguments in favor or against a direct antiviral effect will be discussed. Yet, most patients relapse and alternative therapies are mandatory. IFN and arsenic trioxide induce Tax proteolysis, synergize to induce apoptosis in ATL cells and cure Tax-driven ATL in mice through specific targeting of leukemia initiating cell activity. These results provide a biological basis for the clinical success of arsenic/IFN/AZT therapy in ATL patients and suggest that both extinction of viral replication (AZT) and Tax degradation (arsenic/IFN) are needed to cure ATL.

ATL response to arsenic/interferon therapy is triggered by SUMO/PML/RNF4-dependent Tax degradation

The human T-cell lymphotropic virus type I (HTLV-1) Tax transactivator initiates transformation in adult T-cell leukemia/lymphoma (ATL), a highly aggressive chemotherapy-resistant malignancy. The arsenic/interferon combination, which triggers degradation of the Tax oncoprotein, selectively induces apoptosis of ATL cell lines and has significant clinical activity in Tax-driven murine ATL or human patients. However, the role of Tax loss in ATL response is disputed, and the molecular mechanisms driving degradation remain elusive. Here we demonstrate that ATL-derived or HTLV-1-transformed cells are dependent on continuous Tax expression, suggesting that Tax degradation underlies clinical responses to the arsenic/interferon combination. The latter enforces promyelocytic leukemia protein (PML) nuclear body (NB) formation and partner protein recruitment. In arsenic/interferon-treated HTLV-1 transformed or ATL cells, Tax is recruited onto NBs and undergoes PML-dependent hyper-sumoylation by small ubiquitin-like modifier (SUMO)2/3 but not SUMO1, ubiquitination by RNF4, and proteasome-dependent degradation. Thus, the arsenic/interferon combination clears ATL through degradation of its Tax driver, and this regimen could have broader therapeutic value by promoting degradation of other pathogenic sumoylated proteins.

Animal models on HTLV-1 and related viruses: what did we learn?

Retroviruses are associated with a wide variety of diseases, including immunological, neurological disorders, and different forms of cancer. Among retroviruses, Oncovirinae regroup according to their genetic structure and sequence, several related viruses such as human T-cell lymphotropic viruses types 1 and 2 (HTLV-1 and HTLV-2), simian T cell lymphotropic viruses types 1 and 2 (STLV-1 and STLV-2), and bovine leukemia virus (BLV). As in many diseases, animal models provide a useful tool for the studies of pathogenesis, treatment, and prevention. In the current review, an overview on different animal models used in the study of these viruses will be provided. A specific attention will be given to the HTLV-1 virus which is the causative agent of adult T-cell leukemia/lymphoma (ATL) but also of a number of inflammatory diseases regrouping the HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP), infective dermatitis and some lung inflammatory diseases. Among these models, rabbits, monkeys but also rats provide an excellent in vivo tool for early HTLV-1 viral infection and transmission as well as the induced host immune response against the virus. But ideally, mice remain the most efficient method of studying human afflictions. Genetically altered mice including both transgenic and knockout mice, offer important models to test the role of specific viral and host genes in the development of HTLV-1-associated leukemia. The development of different strains of immunodeficient mice strains (SCID, NOD, and NOG SCID mice) provide a useful and rapid tool of humanized and xenografted mice models, to test new drugs and targeted therapy against HTLV-1-associated leukemia, to identify leukemia stem cells candidates but also to study the innate immunity mediated by the virus. All together, these animal models have revolutionized the biology of retroviruses, their manipulation of host genes and more importantly the potential ways to either prevent their infection or to treat their associated diseases.