Rationale:The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-B (NFB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention.Objective: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. Methods and Results: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2؊/؊ bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2 macrophages. Further analysis in Rip2؊/؊ macrophages showed that the lipid accumulation was scavengerreceptor independent and mediated by Toll-like receptor 4 (TLR4)-dependent lipid uptake. Ⅲ macrophages L ow-density lipoproteins (LDL) are the major extracellular carriers of cholesterol and, as such, play important physiological roles in cellular function and regulation of metabolic pathways. However, under pathological conditions of hyperlipidemia, cholesterol is diverted from its physiological targets and accumulates in lipid-loaded macrophages ("foam cells") in the vascular wall. [1][2][3][4] This pathological deposition of atherogenic lipoproteins activates the inflammatory response that characterizes atherosclerosis. Furthermore, by stimulating the synthesis and secretion of proteoglycans, 5,6 this inflammation further accelerates retention of atherogenic lipoproteins. Thus, lipid accumulation and inflammation are closely linked in atherogenesis. [7][8][9][10][11] The molecular mechanisms that link retention of atherogenic lipoproteins and activation of the inflammatory response are still unclear. Recent evidence implies a key role for the innate immune system and pathogen patternrecognition receptors, in particular the membrane-bound Toll-like receptors (TLRs). 12 Ligand binding to these receptors results in activation of the proinflammatory transcription factor nuclear factor-B (NF-B) and expression of proinflammatory molecules. 12,13 The receptor-interacting protein 2 (Rip2) is a serine/ threonine kinase that activates NF-B and is reported to mediate signaling through both TLRs and Nod-like receptors, although its involvement in TLR signaling has been questioned recently. 14 -18 Furthermore, it was demonstrated recently that the regulation of Rip2 involves a novel feedforward regulatory mechanism: Rip2 not only positively regulates NF-B activity, but inflammatory cytokines that activate the NF-B pathway induce increased Rip2 expression. 19 These studies thus suggest the therapeutic potential of inhibiting Rip2 to inhibit inflammation and...

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSP fused with green fluorescence protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343 base pair (bp) proximal part and the 150 bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. FACS analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150 bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

The hexameric, barrel-forming, AAA+ protease Lon is critical for maintaining mitochondrial matrix protein homeostasis. Efficient substrate processing by Lon requires the coordinated action of six protomers. Despite Lon’s importance for human health, the molecular bases for Lon’s substrate recognition and processing remain unclear. Here, we use a combination of biochemistry and electron cryomicroscopy (cryo-EM) to unveil the structural and functional basis for full-length human mitochondrial Lon’s degradation of mitochondrial transcription factor A (TFAM). We show how opposing protomers in the Lon hexamer barrel interact through their N-terminal domains to give what resembles three feet above the barrel and help to form a triangular pore located just above the entry pore to the barrel. The interactions between opposing protomers constitute a primary allosteric regulation of Lon activity. A secondary allosteric regulation consists of an inter-subunit signaling element in the ATPase domains. By considering the ATP or ADP load in each protomer, we show how this dual allosteric mechanism in Lon achieves coordinated ATP hydrolysis and substrate processing. This mechanism enforces sequential anti-clockwise ATP hydrolysis resulting in a coordinated hand-over-hand translocation of the substrate towards the protease active sites.

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