Fusion crystallization reveals the behavior of both the 1TEL crystallization chaperone and the TNK1 UBA domain.

Human thirty-eight-negative kinase-1 (TNK1) is implicated in cancer progression. The TNK1-UBA domain binds polyubiquitin and plays a regulatory role in TNK1 activity and stability. Sequence analysis suggests an unusual architecture for the TNK1 UBA domain, but an experimentally-validated molecular structure is undetermined. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Å. A 1TEL search model enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its host 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our studies support a mechanism of TELSAM fusion crystallization and show that TELSAM fusion crystals require fewer crystal contacts than traditional protein crystals. Modeling and experimental validation suggest the UBA domain may be selective for both the length and linkages of polyubiquitin chains.

Curcumin modulates multiple cell death, matrix metalloproteinase activation and cardiac protein release in susceptible and resistant Plasmodium berghei-infected mice.

Pro-inflammatory signaling, cell death, and metalloproteinases activation are events in Plasmodium infection. However, it is not known if treatment with mefloquine (MF), and curcumin (CM) supplementation, will modulate these conditions. Malaria was induced in two different studies using susceptible (NK 65, study 1) and resistant (ANKA, study 2) strains of mouse malaria parasites (Plasmodium berghei) in thirty male Swiss mice (n = 5) in each study. Following confirmation of parasitemia, mice received 10 mL/kg distilled water (infected control), MF (10 mg/kg), MF and CM (25 mg/kg), MF and CM (50 mg/kg), CM (25 mg/kg) and CM (50 mg/kg). Five mice (not infected) were used as control. After treatment, the animals were sacrificed, serum obtained and liver mitochondria were isolated. Serum Tumour Necrosis Factor alpha (TNF-α), C-reactive protein (CRP), Interleukins-1 beta (IL-1ß) and Interleukins-6 (IL-6) as well as caspases-3, 9 (C3 and C9), p53, serum troponin I (TI) and creatine kinase (CK), were assayed using ELISA techniques. Mitochondrial membrane permeability transition (mPT) pore opening, mitochondrial F0F1 ATPase activity, and lipid peroxidation (mLPO) were determined spectrophotometrically. Matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) expressions were determined using electrophoresis. CM supplementation (25 mg/kg) significantly decreased serum p53, TNF-α, CRP and IL-6 compared with MF. In the resistant model, CM prevented mPT pore opening, significantly decreased F0F1 ATPase activity and mLPO. MF activated caspase-3 while supplementation with CM significantly decreased this effect. Furthermore, MMP-2 and MMP-9 were selectively expressed in the susceptible model. Malarial treatment with mefloquine elicits different cell death responses while supplementation with curcumin decreased TI level and CK activities.