Development of sensitive cellular assay systems and their application to the identification of "orphan" seven-transmembrane receptors

Seven-transmembrane, G-protein coupled receptors play a central role in physiology since they facilitate cell communication in multicellular organisms by recognition of a broad range of ligands. They also represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and, hence, their physiological functions, remain to be identified. These receptors are usually referred to as “orphan” receptors. A pre-requisite for the identification of ligands activating “orphan” receptors is powerful assay systems displaying a high assay quality as specified by a high Z-value. Until now, reporter gene assays have not been in common use in this process. Therefore, we aimed to develop improved reporter gene assays. This aim was accomplished by optimizing the promoter region of the construct, the reporter enzyme, and the assay procedure. Furthermore, a fluorescence-based clone selection step was introduced, that allowed the selection of the most sensitive reporter cell clones. The established test cell lines responded sensitively upon stimulation of various cell surface receptors as demonstrated by several receptors. In the first approach, transcription of the reporter gene was under control of a synthetic promoter consisting of 9 TPA responsive elements. In a further improved construct, the promoter was extended with six NF-kB and six STAT motifs. The used reporter gene was designed as a fusion gene coding for green fluorescent protein and Photinus luciferase. The amplification of reporter enzyme activity was substantially larger than in any other described system, and the high assay quality made it suitable as a primary screening tool. The development of efficient assays allowed the screening for hitherto unknown ligands to orphan seven-transmembrane receptors. Natural ligands for two recently unknown receptors were identified. Thus, we identified the second leukotriene B4 receptor, BLT2, and the first cell surface, free fatty acid receptor, FFA1. The BLT2 receptor was first identified in silico, cloned, and subsequently functionally expressed in HeLa cells. The identification of FFA1 was accomplished using a reverse pharmacology approach. The FFA1 receptor (previously known as GPR40) responded to medium to long chain free fatty acids, including compounds like ±9-hydroxy-octadecadienoic acid (9-HODE) and a conjugated linoleic acid (10,12-CLA). Receptor expression was detected in heart, skeletal muscle, liver and in pancreatic b-cells. Most importantly, the identification of anti-diabetic drugs (thiazolidinediones and MEDICA16) as agents acting on this receptor implies an important connection between FFA1R and type II diabetes

COX7A2L/SCAFI and Pre-Complex III Modify Respiratory Chain Supercomplex Formation in Different Mouse Strains with a Bcs1/Mutation

The COX7A2L (Supercomplex Assembly Factor I, SCAFI) protein has been proposed to be a mitochondrial supercomplex assembly factor required for respirasome (supercomplex containing complexes I, III, and IV) formation. In the C57BU6 mouse strain a homozygous in-frame 6-base-pair deletion in the COX7a2I/SCAF1 gene resulting in unstable protein and suggesting loss of function was previously identified. The loss of SCAFI was shown to impede respirasome formation, a major concern for the use of C57BL mouse strains in mitochondrial research. In contradiction, another recent study suggested that supercomplex formation is independent of SCAFI isoforms. We investigated whether SCAFI isoform status affected the disease severity and supercomplex formation in the liver of Bcs1a232A>G knock-in mice with incomplete complex III assembly. In homozygotes (Bcs1/(G/G)) of mixed (C57BL/6:129/Sv) genetic background, the lifespan was similar in mice with wild-type SCAFI allele and in those homozygous (SCAF/(short/short)) for the deleted SCAF1 variant (34 3 days; n = 6 vs. 32 +/- 2 days; n = 7, respectively). SCAFI heterozygosity (SCAF/(long/short)) resulted in decreased SCAFI protein but respirasome assembly was unaffected. Congenic (C57BL/6) mice were of the genotype SCAF(short/short) and had no detectable SCAFI protein. In their liver mitochondria, respirasome composition was altered as compared to mixed background mice. Complex IV was mainly present as monomers and dimers, and only low amounts were found in combination with complex I and complex III or with precomplex III. The main supercomplex in the liver mitochondria of C57BU6 mice comprised only complexes I and III. In conclusion, in liver mitochondria of C57BL/6 mice, supercomplexes had markedly reduced amount of, but were not completely depleted of, complex IV, supporting a role for COX7A2L/SCAFI in supercomplex assembly. However, the disease progression of the Bcs1/mutant mice was unrelated to SCAFI isoforms and supercomplex composition, suggesting that other genetic factors contribute to the different survival in the different genetic backgrounds.Peer reviewe

CCR2⁺CD103⁻ intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells

The identification of intestinal macrophages (m phi s) and dendritic cells (DCs) is a matter of intense debate. Although CD103(+) mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103(-) MPs remain controversial. We show here that intestinal CD103(-)CD11b(+) MPs can be separated clearly into DCs and m phi s based on phenotype, gene profile, and kinetics. CD64(-)CD103(-)CD11b(+) MPs are classical DCs, being derived from Flt3 ligand-dependent, DC-committed precursors, not Ly6C hi monocytes. Surprisingly, a significant proportion of these CD103(-)CD11b(+) DCs express CCR2 and there is a selective decrease in CD103(-)CD11b(+) DCs in mice lacking this chemokine receptor. CCR2(+)CD103(-) DCs are present in both the murine and human intestine, drive interleukin (IL)-17a production by Tcells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2(+) DCs that is involved in priming mucosal T helper type 17 (Th17) responses

Effects of Short-term 3-Day Caloric Restriction on Cardiometabolic Health in Overweight and Obese Individuals

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The radical-binding lipocalin A1M binds to a Complex I subunit and protects mitochondrial structure and function.

Aims: During cell death, energy-consuming cell degradation and recycling programs are performed. Maintenance of energy-delivery during cell death is therefore crucial but the mechanisms to keep the mitochondrial functions intact during these processes are poorly understood. We have investigated the hypothesis that the heme- and radical-binding ubiquitous protein A1M (α1-microglobulin) is involved in protection of the mitochondria against oxidative insult during cell death. Results: Using blood cells, keratinocytes and liver cells, we show that A1M binds with high affinity to apoptosis-induced cells and is localized to mitochondria. The mitochondrial Complex I subunit NDUFAB1 was identified as a major molecular target of the A1M-binding. Furthermore, A1M was shown to inhibit the swelling of mitochondria, and to reverse the severely abrogated ATP-production of mitochondria when exposed to heme and ROS. Innovation: Import of the radical- and heme-binding protein A1M from the extracellular compartment confers protection of mitochondrial structure and function during cellular insult. Conclusion: A1M binds to a subunit of Complex I and has a role in assisting the mitochondria to maintain its energy delivery during cell death. A1M may also, at the same time, counteract and eliminate the ROS generated by the mitochondrial respiration to prevent oxidative damage to surrounding healthy tissue

Discovery of TUG-770: a highly potent free fatty acid receptor 1 (FFA1/GPR40) agonist for treatment of type 2 diabetes

Free fatty acid receptor 1 (FFA1 or GPR40) enhances glucose-stimulated insulin secretion from pancreatic β-cells and currently attracts high interest as a new target for the treatment of type 2 diabetes. We here report the discovery of a highly potent FFA1 agonist with favorable physicochemical and pharmacokinetic properties. The compound efficiently normalizes glucose tolerance in diet-induced obese mice, an effect that is fully sustained after 29 days of chronic dosing