The function of Mim1 in the biogenesis of the mitochondrial TOM complex

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into the organelle. The translocase is a multi-subunit complex composed of seven subunits: two receptor proteins, Tom70 and Tom20, and five components which form the core complex, Tom40, Tom22, Tom7, Tom6, and Tom5. In this thesis it is shown that Mim1 is required for the integration of the import receptor Tom20 into the outer membrane but not for its assembly into the TOM complex. Structural characteristics of Mim1 required for its function were studied in detail. Mim1 forms homooligomeric structures via its transmembrane segment which contains two helix-dimerization GXXXG/A motifs. The homooligomerization is a precondition for the function of Mim1 in mediating the integration of Tom20 into the mitochondrial outer membrane

Development of recombinant proteins to treat chronic pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain

No Effects of Hyperosmolar Culture Medium on Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells Despite Upregulation Extracellular Matrix Genes

Study Design. An in vitro study using human degenerated nucleus pulposus cells. Objective. To determine the effect of osmolality and different osmolytes on the regeneration by human nucleus pulposus cells through gene expression and extracellular matrix production. Summary of Background Data. Intervertebral disc (IVD) degeneration is a major problem in developed countries. Regeneration of the IVD can prevent pain and costs due to diminished work absence and health care, and improve quality of life. The osmotic value of a disc decreases during degeneration due to loss of proteoglycans and might increase degeneration. It is known that gene expression of matrix genes of nucleus pulposus (NP) cells increases when cultured in hyperosmotic medium. Thus, increasing the osmolality of the disc might be beneficial for disc regeneration. Methods. In the current study, isolated degenerated human NP cells were used in regeneration culture with medium of different osmolalities, adjusted with different osmolytes. NaCl, urea and sucrose. The cells were cultured for 28 days and expression of matrix genes and production of glycosaminoglycans and collagen II were measured. Results. Gene expression for both collagen II and aggrecan increased with increasing osmolality using NaCl or sucrose, but not urea. Protein production however, was not affected by increasing osmolality and was decreased when using urea and sucrose. Expression of genes for Col1A1, MMP13, and MMP14 decreased with increasing osmolality, whereas expression of LOXL2 and LOXL3 increased. Transient expression of TonEBP was found 6 hours after the start of culture, but not at later time points. Conclusion. Although expression of matrix genes is upregulated, hyperosmolality does not enhance matrix production by nucleus pulposus cells. Raising osmolality can potentially increase matrix production, but in itself is not sufficient to accomplish regeneration in the current in vitro culture system

IL4-10 fusion protein has chondroprotective, anti-inflammatory and potentially analgesic effects in the treatment of osteoarthritis

OBJECTIVE: Effective disease-modifying drugs for osteoarthritis (DMOAD) should preferably have chondroprotective, anti-inflammatory, and analgesic activity combined in a single molecule. We developed a fusion protein of IL4 and IL10 (IL4-10 FP), in which the biological activity of both cytokines is preserved. The present study evaluates the chondroprotective, anti-inflammatory, and analgesic activity of IL4-10 FP in in vitro and in vivo models of osteoarthritis. METHODS: Human osteoarthritic cartilage tissue and synovial tissue were cultured with IL4-10 FP. Cartilage proteoglycan turnover and release of pro-inflammatory, catabolic, and pain mediators by cartilage and synovial tissue were measured. The analgesic effect of intra-articularly injected IL4-10 FP was evaluated in a canine model of osteoarthritis by force-plate analysis. RESULTS: IL4-10 FP increased synthesis (p=0.018) and decreased release (p=0.018) of proteoglycans by osteoarthritic cartilage. Release of pro-inflammatory IL6 and IL8 by cartilage and synovial tissue was reduced in the presence of IL4-10 FP (all p<0.05). The release of MMP3 by osteoarthritic cartilage and synovial tissue was decreased (p=0.018 and 0.028) whereas TIMP1 production was not significantly changed. Furthermore, IL4-10 FP protected cartilage against destructive properties of synovial tissue mediators shown by the increased cartilage proteoglycan synthesis (p=0.0235) and reduced proteoglycan release (p=0.0163). Finally, intra-articular injection of IL4-10 FP improved the deficient joint loading in dogs with experimentally induced osteoarthritis. CONCLUSION: The results of current preliminary study suggest that IL4-10 FP has DMOAD potentials since it shows chondroprotective and anti-inflammatory effects in vitro, as well as potentially analgesic effect in a canine in vivo model of osteoarthritis

IL4-10 fusion protein has chondroprotective, anti-inflammatory and potentially analgesic effects in the treatment of osteoarthritis

OBJECTIVE: Effective disease-modifying drugs for osteoarthritis (DMOAD) should preferably have chondroprotective, anti-inflammatory, and analgesic activity combined in a single molecule. We developed a fusion protein of IL4 and IL10 (IL4-10 FP), in which the biological activity of both cytokines is preserved. The present study evaluates the chondroprotective, anti-inflammatory, and analgesic activity of IL4-10 FP in in vitro and in vivo models of osteoarthritis. METHODS: Human osteoarthritic cartilage tissue and synovial tissue were cultured with IL4-10 FP. Cartilage proteoglycan turnover and release of pro-inflammatory, catabolic, and pain mediators by cartilage and synovial tissue were measured. The analgesic effect of intra-articularly injected IL4-10 FP was evaluated in a canine model of osteoarthritis by force-plate analysis. RESULTS: IL4-10 FP increased synthesis (p=0.018) and decreased release (p=0.018) of proteoglycans by osteoarthritic cartilage. Release of pro-inflammatory IL6 and IL8 by cartilage and synovial tissue was reduced in the presence of IL4-10 FP (all p<0.05). The release of MMP3 by osteoarthritic cartilage and synovial tissue was decreased (p=0.018 and 0.028) whereas TIMP1 production was not significantly changed. Furthermore, IL4-10 FP protected cartilage against destructive properties of synovial tissue mediators shown by the increased cartilage proteoglycan synthesis (p=0.0235) and reduced proteoglycan release (p=0.0163). Finally, intra-articular injection of IL4-10 FP improved the deficient joint loading in dogs with experimentally induced osteoarthritis. CONCLUSION: The results of current preliminary study suggest that IL4-10 FP has DMOAD potentials since it shows chondroprotective and anti-inflammatory effects in vitro, as well as potentially analgesic effect in a canine in vivo model of osteoarthritis

Sialic Acid-Engineered IL4-10 Fusion Protein is Bioactive and Rapidly Cleared from the Circulation

PURPOSE: Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4-10 fusion protein (IL4-10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. METHODS: CHO cell lines producing low (IL4-10 FP lowSA) and high sialylated (IL4-10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4-10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. RESULTS: IL4-10 FP lowSA and IL4-10 FP highSA had similar potency in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4-10 FP lowSA, whereas the calculated half-life of both IL4-10 FP lowSA and IL4-10 FP highSA was 20.7 min. Finally, both IL4-10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. CONCLUSIONS: Differential sialylation of IL4-10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4-10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment

Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain

Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain