Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles.

TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein. We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased. Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax ∼95 Å in DPC alone versus ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (Kd ∼70 µM in DPC alone versus ∼0.91 µM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST). In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.

Elucidating the pivotal role of TSPO in porphyrin-related cellular processes, in Bacillus cereus.

A structural homolog of the mammalian TSPO has been identified in the human pathogen Bacillus cereus. BcTSPO, in its recombinant form, has previously been shown to bind and degrade porphyrins. In this study, we generated a ΔtspO mutant strain in B. cereus ATCC 14579 and assessed the impact of the absence of BcTSPO on cellular proteomics and physiological characteristics. The proteomic analysis revealed correlations between the lack of BcTSPO and the observed growth defects, increased oxygen consumption, ATP deficiency, heightened tryptophan catabolism, reduced motility, and impaired biofilm formation in the ΔtspO mutant strain. Our results also suggested that BcTSPO plays a crucial role in regulating intracellular levels of metabolites from the coproporphyrin-dependent branch of the heme biosynthetic pathway. This regulation potentially underlies alterations in the metabolic landscape, emphasizing the pivotal role of BcTSPO in B. cereus aerobic metabolism. Notably, our study unveils, for the first time, the involvement of TSPO in tryptophan metabolism. These findings underscore the multifaceted role of TSPO, not only in metabolic pathways but also potentially in the microorganism's virulence mechanisms.

Among the recombinant TSPOs, the BcTSPO.

Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion bodies (IB). Unlike mouse TSPO, BcTSPO purified in SDS detergent from IB is well structured and can bind various ligands such as high-affinity PK 11195, protoporphyrin IX (PPIX) and δ-aminolevulinic acid (ALA). For each of the three ligands, 1H-15N HSQC titration NMR experiments suggest that different amino acids of BcTSPO binding cavity are involved in the interaction. PPIX, an intermediate of heme biosynthesis, binds to the cavity of BcTSPO and its fluorescence can be significantly reduced in the presence of light and oxygen. The light irradiation leads to two products that have been isolated and characterized as photoporphyrins. They result from the addition of singlet oxygen to the two vinyl groups hence leading to the formation of hydroxyaldehydes. The involvement of water molecules, recently observed along with the binding of heme in Rhodobacter sphaeroides (RsTSPO) is highly probable. Altogether, these results raise the question of the role of TSPO in heme biosynthesis regulation as a possible scavenger of reactive intermediates.

Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein.

The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation. Here, we decipher the solution structures of the recombinant mTSPO without ligand both in (i) SDS, the detergent used to extract and purify the protein from E. coli inclusion bodies, and (ii) DPC, the detergent used to solve the PK 11195-binding mTSPO NMR structure. We report partially refolded and less flexible mTSPO helices in DPC compared to SDS. Besides, DPC stabilizes the tertiary structure of mTSPO, as shown by a higher intrinsic Trp fluorescence and changes in indole environment. We evaluate by SEC-MALLS that ∼135 SDS and ∼100 DPC molecules are bound to mTSPO. SEC-small-angle X-ray (SAXS) and neutron (SANS) scattering confirm a larger mTSPO-detergent complex in SDS than in DPC. Using the contrast-matching technique in SEC-SANS, we demonstrate that mTSPO conformation is more compact and less flexible in DPC than in SDS. Combining ab initio modeling with SANS, we confirm that mTSPO conformation is less elongated in DPC than in SDS. However, the free-ligand mTSPO envelope in DPC is not as compact as the PK 11195-binding protein NMR structure, the ligand stiffening the protein.

Solid-state NMR study of structural heterogeneity of the apo WT mouse TSPO reconstituted in liposomes.

In the last decades, ligand binding to human TSPO has been largely used in clinical neuroimaging, but little is known about the interaction mechanism. Protein conformational mobility plays a key role in the ligand recognition and both, ligand-free and ligand-bound structures, are mandatory for characterizing the molecular binding mechanism. In the absence of crystals for mammalian TSPO, we have exploited solid-state nuclear magnetic resonance (ssNMR) spectroscopy under magic-angle spinning (MAS) to study the apo form of recombinant mouse TSPO (mTSPO) reconstituted in lipids. This environment has been previously described to permit binding of its high-affinity drug ligand PK11195 and appears therefore favourable for the study of molecular dynamics. We have optimized the physical conditions to get the best resolution for MAS ssNMR spectra of the ligand-free mTSPO. We have compared and combined various ssNMR spectra to get dynamical information either for the lipids or for the mTSPO. Partial assignment of residue types suggests few agreements with the published solution NMR assignment of the PK11195-bound mTSPO in DPC detergent. Moreover, we were able to observe some lateral chains of aromatic residues that were not assigned in solution. 13C double-quantum NMR spectroscopy shows remarkable dynamics for ligand-free mTSPO in lipids which may have significant implications on the recognition of the ligand and/or other protein partners.

Targeting hIAPP fibrillation: A new paradigm to prevent ß-cell death?

Loss of pancreatic ß-cell mass is deleterious for type 2 diabetes patients since it reduces insulin production, critical for glucose homeostasis. The main research axis developed over the last few years was to generate new pancreatic ß-cells or to transplant pancreatic islets as occurring for some specific type 1 diabetes patients. We evaluate here a new paradigm consisting in preservation of ß-cells by prevention of human islet amyloid polypeptide (hIAPP) oligomers and fibrils formation leading to pancreatic ß-cell death. We review the hIAPP physiology and the pathology that contributes to ß-cell destruction, deciphering the various cellular steps that could be involved. Recent progress in understanding other amyloidosis such as Aß, Tau, α-synuclein or prion, involved in neurodegenerative processes linked with inflammation, has opened new research lines of investigations to preserve neuronal cells. We evaluate and estimate their transposition to the pancreatic ß-cells preservation. Among them is the control of reactive oxygen species (ROS) production occurring with inflammation and the possible implication of the mitochondrial translocator protein as a diagnostic and therapeutic target. The present review also focuses on other amyloid forming proteins from molecular to physiological and physiopathological points of view that could help to better decipher hIAPP-induced ß-cell death mechanisms and to prevent hIAPP fibril formation.

CX3CL1 homo-oligomerization drives cell-to-cell adherence.

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.

A Plant-Specific N-terminal Extension Reveals Evolutionary Functional Divergence within Translocator Proteins.

Conserved translocator proteins (TSPOs) mediate cell stress responses possibly in a cell-type-specific manner. This work reports on the molecular function of plant TSPO and their possible evolutionary divergence. Arabidopsis thaliana TSPO (AtTSPO) is stress induced and has a conserved polybasic, plant-specific N-terminal extension. AtTSPO reduces water loss by depleting aquaporin PIP2;7 in the plasma membrane. Herein, AtTSPO was found to bind phosphoinositides in vitro, but only full-length AtTSPO or chimeric mouse TSPO with an AtTSPO N-terminus bound PI(4,5)P2in vitro and modified PIP2;7 levels in vivo. Expression of AtTSPO but not its N-terminally truncated variant enhanced phospholipase C activity and depleted PI(4,5)P2 from the plasma membrane and its enrichment in Golgi membranes. Deletion or point mutations within the AtTSPO N-terminus affected PI(4,5)P2 binding and almost prevented AtTSPO-PIP2;7 interaction in vivo. The findings imply functional divergence of plant TSPOs from bacterial and animal counterparts via evolutionary acquisition of the phospholipid-interacting N-terminus.

TSPO2 translocates 5-aminolevulinic acid into human erythroleukemia cells.

BACKGROUND: 5-Aminolevulinic acid (ALA) is the first precursor of heme biosynthesis pathway. The exogenous addition of ALA to cells leads to protoporphyrin IX (PPIX) accumulation that has been exploited in photodynamic diagnostic and photodynamic therapy. Several types of ALA transporters have been described depending on the cell type, but there was no clear entry pathway for erythroid cells. The 18 kDa translocator protein (TSPO) has been proposed to be involved in the transport of porphyrins and heme analogs. RESULTS: ALA-induced PPIX accumulation in erythroleukemia cells (UT-7 and K562) was impaired by PK 11195, a competitive inhibitor of both transmembrane proteins TSPO (1 and 2). PK 11195 did not modify the activity of the enzymes of heme biosynthesis, suggesting that ALA entry at the plasma membrane was the limiting factor. In contrast, porphobilinogen (PBG)-induced PPIX accumulation was not affected by PK 11195, suggesting that plasma membrane TSPO2 is a selective transporter of ALA. Overexpression of TSPO2 at the plasma membrane of erythroleukemia cells increased ALA-induced PPIX accumulation, confirming the role of TSPO2 in the import of ALA into the cells. CONCLUSIONS: ALA-induced PPIX accumulation in erythroid cells involves TSPO2 as a selective translocator through the plasma membrane. SIGNIFICANCE: This is the first characterisation of molecular mechanisms involving a new actor in ALA transport in ALA-induced PPIX accumulation in erythroleukemia cells, which could be inhibited by specific drug ligands.

Concentration, distribution, and influence of aging on the 18 kDa translocator protein in human brain: Implications for brain imaging studies.

Positron emission tomography (PET) imaging of the translocator protein (TSPO) is widely used as a biomarker of microglial activation. However, TSPO protein concentration in human brain has not been optimally quantified nor has its regional distribution been compared to TSPO binding. We determined TSPO protein concentration, change with age, and regional distribution by quantitative immunoblotting in autopsied human brain. Brain TSPO protein concentration (>0.1 ng/µg protein) was higher than those reported by in vitro binding assays by at least 2 to 70 fold. TSPO protein distributed widely in both gray and white matter regions, with distribution in major gray matter areas ranked generally similar to that of PET binding in second-generation radiotracer studies. TSPO protein concentration in frontal cortex was high at birth, declined precipitously during the first three months, and increased modestly during adulthood/senescence (10%/decade; vs. 30% for comparison astrocytic marker GFAP). As expected, TSPO protein levels were significantly increased (+114%) in degenerating putamen in multiple system atrophy, providing further circumstantial support for TSPO as a gliosis marker. Overall, findings show some similarities between TSPO protein and PET binding characteristics in the human brain but also suggest that part of the TSPO protein pool might be less available for radioligand binding.

Insight into the Structural Features of TSPO: Implications for Drug Development.

The translocator protein (TSPO), an 18-kDa transmembrane protein primarily found in the outer mitochondrial membrane, is evolutionarily conserved and widely distributed across species. In mammals, TSPO has been described as a key member of a multiprotein complex involved in many putative functions and, over the years, several classes of ligand have been developed to modulate these functions. In this review, we consider the currently available atomic structures of mouse and bacterial TSPO and propose a rationale for the development of new ligands for the protein. We provide a review of TSPO monomeric and oligomeric states and their conformational flexibility, together with ligand-binding site and interaction mechanisms. These data are expected to help considerably the development of high-affinity ligands for TSPO-based therapies or diagnostics.

Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO.

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

Erythroid-Progenitor-Targeted Gene Therapy Using Bifunctional TFR1 Ligand-Peptides in Human Erythropoietic Protoporphyria.

Erythropoietic protoporphyria (EPP) is a hereditary disease characterized by a deficiency in ferrochelatase (FECH) activity. FECH activity is responsible for the accumulation of protoporphyrin IX (PPIX). Without etiopathogenic treatment, EPP manifests as severe photosensitivity. 95% of affected individuals present a hypomorphic FECH allele trans to a loss-of-function (LOF) FECH mutation, resulting in a reduction in FECH activity in erythroblasts below a critical threshold. The hypomorphic allele promotes the use of a cryptic acceptor splice site, generating an aberrant FECH mRNA, which is responsible for the reduced level of wild-type FECH mRNA and, ultimately, FECH activity. We have previously identified an antisense oligonucleotide (AON), AON-V1 (V1), that redirects splicing to the physiological acceptor site and reduces the accumulation of PPIX. Here, we developed a specific strategy that uses transferrin receptor 1 (TRF1) as a Trojan horse to deliver V1 to erythroid progenitors. We designed a bifunctional peptide (P1-9R) including a TFR1-targeting peptide coupled to a nine-arginine cell-penetrating peptide (CPP) that facilitates the release of the AON from TFR1 in endosomal vesicles. We demonstrated that the P1-9R/V1 nanocomplex promotes the efficient and prolonged redirection of splicing towards the physiological splice site and subsequent normalization of WT FECH mRNA and protein levels. Finally, the P1-9R/V1 nanocomplex increases WT FECH mRNA production and significantly decreases PPIX accumulation in primary cultures of differentiating erythroid progenitors from an overt EPP-affected individual. P1-9R is a method designed to target erythroid progenitors and represents a potentially powerful tool for the in vivo delivery of therapeutic DNA in many erythroid disorders.

Recombinant Overexpression of Mammalian TSPO Isoforms 1 and 2.

TSPO is a 18 kDa membrane protein that exists in mammalian as two isoforms 1 and 2. They are involved in different functions and are located in different membranes. TSPO1 is mainly located in outer mitochondrial membrane, whereas TSPO2 is encountered in plasma membrane of red blood cells. Determination of their structures is a milestone to understand their function. Their natural abundance is not sufficient to get large amounts usually required for structural studies. We described heterologous overexpression in both bacterial and cell-free system and purification on immobilized-metal affinity chromatography (IMAC) of both proteins. Using the same vector, TSPO1 is mostly recovered in bacterial inclusion bodies whereas TSPO2 is found in both bacterial cytosol and inclusion bodies, but in low amounts. Cell-free expression was the best system to overexpress pure TSPO2.

Solid-State NMR of Membrane Protein Reconstituted in Proteoliposomes, the Case of TSPO.

Structural studies of membrane proteins (MP) in a native or native-like environment remain a challenge. X-ray crystallography of three-dimensional crystals of MP in lipids and cryo-electron microscopy of two-dimensional crystals also in lipids have given atomic structures of several MP. Recent developments of solid-state NMR (ssNMR) provided structural data of MP in lipids and should give access to the dynamic behavior of MP's in a native-like environment. Preparation of samples for ssNMR is not trivial with overexpressed proteins since purified recombinant MP have to be reincorporated in proteoliposomes and concentrated in the small volume of the rotor used for ssNMR studies. We present here the protocol that we have used to study the recombinant mouse TSPO1, an integral membrane protein of 20 kDa mostly found in the outer membrane of mitochondria and overexpressed in E. coli bacteria.

A Central Cysteine Residue Is Essential for the Thermal Stability and Function of SUMO-1 Protein and SUMO-1 Peptide-Protein Conjugates.

SUMOylation constitutes a major post-translational modification (PTM) used by the eukaryote cellular machinery to modulate protein interactions of the targeted proteins. The small ubiquitin-like modifier-1 (SUMO-1) features a central and conserved cysteine residue (Cys52) that is located in the hydrophobic core of the protein and in tight contact with Phe65, suggesting the occurrence of an S/π interaction. To investigate the importance of Cys52 on SUMO-1 thermal stability and biochemical properties, we produced by total chemical synthesis SUMO-1 or SUMO-1 Cys52Ala peptide-protein conjugates featuring a native isopeptidic bond between SUMO-1 and a peptide derived from p53 tumor suppressor protein. The Cys52Ala modification perturbed SUMO-1 secondary structure and resulted in a dramatic loss of protein thermal stability. Moreover, the cleavage of the isopeptidic bond by the deconjugating enzyme Upl1 was significantly less efficient than for the wild-type conjugate. Similarly, the in vitro SUMOylation of RanGap1 by E1/E2 conjugating enzymes was significantly less efficient with the SUMO-1 C52A analog compared to wild-type SUMO-1. These data demonstrate the critical role of Cys52 in maintaining SUMO-1 conformation and function and the importance of keeping this cysteine intact for the study of SUMO-1 protein conjugates.

Translocator Protein-Mediated Stabilization of Mitochondrial Architecture during Inflammation Stress in Colonic Cells.

UNLABELLED: Chronic inflammation of the gastrointestinal tract increasing the risk of cancer has been described to be linked to the high expression of the mitochondrial translocator protein (18 kDa; TSPO). Accordingly, TSPO drug ligands have been shown to regulate cytokine production and to improve tissue reconstruction. We used HT-29 human colon carcinoma cells to evaluate the role of TSPO and its drug ligands in tumor necrosis factor (TNF)-induced inflammation. TNF-induced interleukin (IL)-8 expression, coupled to reactive oxygen species (ROS) production, was followed by TSPO overexpression. TNF also destabilized mitochondrial ultrastructure, inducing cell death by apoptosis. Treatment with the TSPO drug ligand PK 11195 maintained the mitochondrial ultrastructure, reducing IL-8 and ROS production and cell death. TSPO silencing and overexpression studies demonstrated that the presence of TSPO is essential to control IL-8 and ROS production, so as to maintain mitochondrial ultrastructure and to prevent cell death. Taken together, our data indicate that inflammation results in the disruption of mitochondrial complexes containing TSPO, leading to cell death and epithelia disruption. SIGNIFICANCE: This work implicates TSPO in the maintenance of mitochondrial membrane integrity and in the control of mitochondrial ROS production, ultimately favoring tissue regeneration.

Probing the gel to liquid-crystalline phase transition and relevant conformation changes in liposomes by (13)C magic-angle spinning NMR spectroscopy.

A straightforward way to visualize gel to liquid-crystalline phase transition in phospholipid membranes is presented by using ¹³C magic-angle spinning NMR. The changes in the 13C isotropic chemical shifts with increasing temperature are shown to be a sensitive probe of the main thermotropic phase transition related to lipid hydrocarbon chain dynamics and relevant conformational changes. The average value of the energy difference between trans and gauche states in the central C4­11 fragment of the DMPC acyl chain was estimated to be 4.02 ± 0.2 kJ mol⁻¹ in the liquid crystalline phase. The reported spectral features will be useful in 13C solid state NMR studies for direct monitoring of the effective lipid chain melting allowing rapid uniaxial rotation of membrane proteins in the biologically relevant liquid-crystalline phase.

Construction and validation of an atomic model for bacterial TSPO from electron microscopy density, evolutionary constraints, and biochemical and biophysical data.

The 18 kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications.

Genetic variation at KIT locus may predispose to melanoma.

As loss of KIT frequently occurs in melanoma progression, we hypothesized that KIT is implicated in predisposition to melanoma (MM). Thus, we sequenced the KIT coding region in 112 familial MM cases and 143 matched controls and genotyped tag single-nucleotide polymorphisms (SNPs) in two cohorts of melanoma patients and matched controls. Five rare KIT substitutions, all predicted possibly or probably deleterious, were identified in five patients, but none in controls [RR = 2.26 (1.26-2.26)]. Expressed in melanocyte lines, three substitutions inhibited KIT signaling. Comparison with exomes database (7020 alleles) confirmed a significant excess of rare deleterious KIT substitutions in patients. Additionally, a common SNP, rs2237028, was associated with MM risk, and 6 KIT variants were associated with nevus count. Our data strongly suggest that rare KIT substitutions predispose to melanoma and that common variants at KIT locus may also impact nevus count and melanoma risk.