Nuno Saraiva

Six-transmembrane Topology for Golgi Anti-apoptotic Protein (GAAP) and Bax Inhibitor 1 (BI-1) Provides Model for the Transmembrane Bax Inhibitor-containing Motif (TMBIM) Family

Background: Golgi anti-apoptotic protein (GAAP) is a regulator of intracellular Ca2+ fluxes and apoptosis. Results: The transmembrane topology of viral GAAP is conserved in human GAAP and BI-1. Conclusion: GAAPs and BI-1 have a six membrane-spanning topology with cytosolic N and C termini and a C-terminal reentrant loop. Significance: The topology of the TMBIM family provides valuable structural information on these proteins. The Golgi anti-apoptotic protein (GAAP) is a hydrophobic Golgi protein that regulates intracellular calcium fluxes and apoptosis. GAAP is highly conserved throughout eukaryotes and some strains of vaccinia virus (VACV) and camelpox virus. Based on sequence, phylogeny, and hydrophobicity, GAAPs were classified within the transmembrane Bax inhibitor-containing motif (TMBIM) family. TMBIM members are anti-apoptotic and were predicted to have seven-transmembrane domains (TMDs). However, topology prediction programs are inconsistent and predicted that GAAP and other TMBIM members have six or seven TMDs. To address this discrepancy, we mapped the transmembrane topology of viral (vGAAP) and human (hGAAP), as well as Bax inhibitor (BI-1). Data presented show a six-, not seven-, transmembrane topology for vGAAP with a putative reentrant loop at the C terminus and both termini located in the cytosol. We find that this topology is also conserved in hGAAP and BI-1. This places the charged C terminus in the cytosol, and mutation of these charged residues in hGAAP ablated its anti-apoptotic function. Given the highly conserved hydrophobicity profile within the TMBIM family and recent phylogenetic data indicating that a GAAP-like protein may have been the ancestral progenitor of a subset of the TMBIM family, we propose that this vGAAP topology may be used as a model for the remainder of the TMBIM family of proteins. The topology described provides valuable information on the structure and function of an important but poorly understood family of proteins.

hGAAP promotes cell adhesion and migration via the stimulation of store-operated Ca2+ entry and calpain 2.

hGAAP promotes cell adhesion and migration by increasing localized Ca2+-dependent activation of calpain, leading to increased focal adhesion dynamics.

Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium

Background: GAAPs are Golgi anti-apoptotic proteins that modulate Ca2+ signaling. Results: Unlike human GAAP, viral GAAP oligomerization is cysteine-dependent. Monomeric vGAAP is functional. Conclusion: Viral and human GAAPs show pH-dependent oligomerization via different mechanisms. Monomeric vGAAP is anti-apoptotic and modulates intracellular Ca2+. Significance: This is the first mechanistic analysis of TMBIM protein oligomerization, and shows that monomers are functional. Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca2+ content of intracellular stores, and regulate Ca2+ fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca2+-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca2+ stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

Ochratoxin A-induced cytotoxicity, genotoxicity and reactive oxygen species in kidney cells: An integrative approach of complementary endpoints.

Ochratoxin A (OTA) is a well-known nephrotoxic and potential carcinogenic agent but no consensus about the molecular mechanisms underlying its deleterious effects has been reached yet. The aim of this study is to integrate several endpoints concerning OTA-induced toxicological effects in Vero kidney cells in order to obtain additional mechanistic data, especially regarding the influence of reactive oxygen species (ROS). One innovative aspect of this work is the use of the superoxide dismutase mimic (SODm) MnTnHex-2-PyP as a mechanistic tool to clarify the involvement of oxidative stress in OTA toxicity. The results showed concentration and time-dependent cytotoxic effects of OTA (crystal violet, neutral red and LDH leakage assays). While the SODm mildly increased cell viability, trolox and ascorbic acid had no effect with regards to this endpoint. OTA induced micronuclei formation. Using the FPG modified comet assay, OTA modestly increased the % of DNA in tail, revealing the presence of oxidative DNA lesions. This mycotoxin increased apoptosis, which was attenuated by SODm. In addition, the SODm decreased the ROS accumulation observed in DHE assay. Taken together, our data suggest that ROS partially contribute to the cytotoxicity and genotoxicity of OTA, although other mechanisms may be relevant in OTA-induced deleterious effects.

Golgi anti-apoptotic proteins are highly conserved ion channels that affect apoptosis and cell migration.

Background: GAAPs regulate intracellular Ca2+ fluxes, cell migration, and apoptosis. Results: GAAP forms a cation-selective channel, and residues involved in its ion-conducting properties were identified. Conclusion: Mutations within the pore demonstrate that GAAP effects on apoptosis and migration are separable. Significance: Characterization of the pore region of GAAP provides insight into the mechanism of action of this novel and highly conserved ion channel. Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca2+ content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

Role of the Copper(II) Complex Cu[15]pyN5 in Intracellular ROS and Breast Cancer Cell Motility and Invasion.

Multiple mechanisms related to metastases undergo redox regulation. Cu[15]pyN5 is a redox‐active copper(II) complex previously studied as a chemotherapy sensitizer in mammary cells. The effects of a cotreatment with Cu[15]pyN5 and doxorubicin (dox) were evaluated in two human breast cancer cell lines: MCF7 (low aggressiveness) and MDA‐MB‐231 (highly aggressive). Cu[15]pyN5 decreased MCF7‐directed cell migration. In addition, a cotreatment with dox and Cu[15]pyN5 reduced the proteolytic invasion of MDA‐MB‐231 cells. Cell detachment was not affected by exposure to these agents. Cu[15]pyN5 and dox significantly increased intracellular ROS in both cell lines. This increase could be at least partially due to H2O2 accumulation. The combination of Cu[15]pyN5 with dox may be beneficial in breast cancer treatment as it could help reduce cancer cell migration and invasion. Moreover, the ligand [15]pyN5 has a high affinity for copper(II) and displays potential anti‐angiogenic properties. Overall, we present a potential drug that might arrest the progression of breast cancer by different and complementary mechanisms.

HIV-1 Vif Interaction with APOBEC3 Deaminases and its Characterization by a New Sensitive Assay

The human APOBEC3 (A3) cytidine deaminases, such as APOBEC3G (A3G) and APOBEC3F (A3F), are potent inhibitors of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif (viral infectivity factor) binds A3 proteins and targets these proteins for ubiquitination and proteasomal degradation. As such, the therapeutic blockage of Vif–A3 interaction is predicted to stimulate natural antiviral activity by rescuing APOBEC expression and virion packaging. In this study, we describe a successful application of the Protein Fragment Complementation Assay (PCA) based on the enzyme TEM-1 β-lactamase to study Vif–A3 interactions. PCA is based on the interaction between two protein binding partners (e.g., Vif and A3G), which are fused to the two halves of a dissected marker protein (β-lactamase). Binding of the two partners reassembles β-lactamase and hence reconstitutes its activity. To validate our assay, we studied the effect of well-described Vif (DRMR, YRHHY) and A3G (D128K) mutations on the interaction between the two proteins. Additionally, we studied the interaction of human Vif with other members of the A3 family: A3F and APOBEC3C (A3C). Our results demonstrate the applicability of PCA as a simple and reliable technique for the assessment of Vif–A3 interactions. Furthermore, when compared with co-immunoprecipitation assays, PCA appeared to be a more sensitive technique for the quantitative assessment of Vif–A3 interactions. Thus, with our results, we conclude that PCA could be used to quantitatively study specific domains that may be involved in the interaction between Vif and APOBEC proteins.

Golgi anti-apoptotic protein: A tale of camels, calcium, channels and cancer

Golgi anti-apoptotic protein (GAAP), also known as transmembrane Bax inhibitor-1 motif-containing 4 (TMBIM4) or Lifeguard 4 (Lfg4), shares remarkable amino acid conservation with orthologues throughout eukaryotes, prokaryotes and some orthopoxviruses, suggesting a highly conserved function. GAAPs regulate Ca2+ levels and fluxes from the Golgi and endoplasmic reticulum, confer resistance to a broad range of apoptotic stimuli, promote cell adhesion and migration via the activation of store-operated Ca2+ entry, are essential for the viability of human cells, and affect orthopoxvirus virulence. GAAPs are oligomeric, multi-transmembrane proteins that are resident in Golgi membranes and form cation-selective ion channels that may explain the multiple functions of these proteins. Residues contributing to the ion-conducting pore have been defined and provide the first clues about the mechanistic link between these very different functions of GAAP. Although GAAPs are naturally oligomeric, they can also function as monomers, a feature that distinguishes them from other virus-encoded ion channels that must oligomerize for function. This review summarizes the known functions of GAAPs and discusses their potential importance in disease.

Choline- versus imidazole-based ionic liquids as functional ingredients in topical delivery systems: cytotoxicity, solubility, and skin permeation studies

Abstract Background: Poor drug solubility represents a problem for the development of topical formulations. Since ionic liquids (ILs) can be placed in either lipophilic or hydrophilic solutions, they may be advantageous vehicles in such delivery systems. Nonetheless, it is vital to determine their usefulness when used at concentrations were cell viability is maintained, which was considered herein. Method: Five different ILs were prepared—three imidazole-based ILs: [C2mim][Br], [C4mim][Br], and [C6mim][Br]; and two choline-based ILs: [Cho][Phe] and [Cho][Glu]. Their cytotoxicity in human keratinocytes (HaCat cells), their influence in drug solubility and in percutaneous permeation, using pig skin membranes, was evaluated. Results: Caffeine and salicylic acid were used as model actives. Choline-based ILs proved to be more suitable as functional ingredients, since they showed higher impact on drug solubility and a lower cytotoxicity. The major solubility enhancement was observed for caffeine and further solubility studies were carried out with this active in several concentrations of the choline-based ILs (0.1; 0.2; 0.5; 1.0; 3.0 and 5.0%, w/w) at 25 °C and 32 °C. Solubility was greatly influenced by concentrations up to 0.5%. The choline-based ILs showed no significant impact on the skin permeation, for both actives. The size of the imidazole-based ILs alkyl chain enhances the caffeine solubility and permeation, but also the ILs cytotoxicity. Stable O/W emulsions and gels were prepared containing the less toxic choline-based ILs and caffeine. Conclusions: Our results indicate that the choline-based ILs were effective functional ingredients, since, when used at nontoxic concentrations, they allowed a higher drug loading, while maintaining the stability of the formulations.

The APE1 redox inhibitor E3330 reduces collective cell migration of human breast cancer cells and decreases chemoinvasion and colony formation when combined with docetaxel

The human apurinic/apyrimidinic endonuclease 1 (APE1) is an ubiquitous multifunctional DNA repair enzyme and a redox signalling protein. Our work addressed the inhibition of APE1 redox function using E3330, as single agent or in combination with docetaxel (DTX), in human breast cancer MDA‐MB‐231 cells. E3330 decreased the colony formation of DTX‐treated cells. In addition, E3330 alone significantly reduced the collective cell migration as assessed by the wound‐healing assay, whereas the combined treatment decreased chemoinvasion. These results suggest that the inhibition of APE1 redox function might have therapeutic potential by modulating cell migration and invasion in metastatic breast cancer.