Multistep phosphorylation systems: tunable components of biological signaling circuits

Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase–dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications.     

Community-level education accelerates the cultural evolution of fertility decline

Explaining why fertility declines as populations modernize is a profound theoretical challenge. It remains unclear whether the fundamental drivers are economic or cultural in nature. Cultural evolutionary theory suggests that community-level characteristics, for example average education, can alter how low-fertility preferences are transmitted and adopted. These assumptions have not been empirically tested. Here, we show that community-level education accelerates fertility decline in a way that is neither predicted by individual characteristics, nor by the level of economic modernization in a population. In 22 high-fertility communities in Poland, fertility converged on a smaller family size as average education in the community increased—indeed community-level education had a larger impact on fertility decline than did individual education. This convergence was not driven by educational levels being more homogeneous, but by less educated women having fewer children than expected, and more highly educated social networks, when living among more highly educated neighbours. The average level of education in a community may influence the social partners women interact with, both within and beyond their immediate social environments, altering the reproductive norms they are exposed to. Given a critical mass of highly educated women, less educated neighbours may adopt their reproductive behaviour, accelerating the pace of demographic transition. Individual characteristics alone cannot capture these dynamics and studies relying solely on them may systematically underestimate the importance of cultural transmission in driving fertility declines. Our results are inconsistent with a purely individualistic, rational-actor model of fertility decline and suggest that optimization of reproduction is partly driven by cultural dynamics beyond the individual.     

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

Human OX₁ orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB₁ cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX₁, OX₂ and CB₁ receptors, C-terminally fused with either Renilla luciferase or GFP² green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB₁ receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP² to CB₁ produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX₁–OX₂ interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB₁ receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.     

The SigB sigma factor mediates high-temperature responses in the cyanobacterium Synechocystis sp. PCC6803

The sigma factors of RNA polymerase play central roles when bacteria adapt to different environmental conditions. We studied heat-shock responses in the cyanobacterium Synechocystis sp. PCC6803 using the sigma factor inactivation strains deltasigB, deltasigD and deltasigBD. The SigB factor was found to be important for short-term heat-shock responses and acquired thermotolerance. The normal high-temperature induction of the hspA gene depended on the SigB factor. The SigD sigma factor had a role in high-temperature responses as well, and the double inactivation strain deltasigBD grew more slowly at 43 degrees C than the deltasigB and deltasigD strains.     

Hexose transporters GLUT1 and GLUT3 are colocalized with hexokinase I in caveolae microdomains of rat spermatogenic cells

Postmeiotic spermatogenic cells, but not meiotic spermatogenic cells respond differentially with glucose-induced changes in [Ca2+]i indicating a differential transport of glucose via facilitative hexose transporters (GLUTs) specifically distributed in the plasma membrane. Several studies have indicated that plasma membrane in mammalian cells is not homogeneously organized, but contains specific microdomains known as detergent-resistant membrane domains (DRMDs), lipid rafts or caveolae. The association of these domains and GLUTs isoforms has not been characterized in spermatogenic cells. We analyzed the expression and function of GLUT1 and GLUT3 in isolated spermatocytes and spermatids. The results showed that spermatogenic cells express both glucose transporters, with spermatids exhibiting a higher affinity glucose transport system. In addition, spermatogenic cells express caveolin-1, and glucose transporters colocalize with caveolin-1 in caveolin-enriched membrane fractions. Experiments in which the integrity of caveolae was disrupted by pretreatment with methyl-beta-cyclodextrin, indicated that the involvement of cholesterol-enriched plasma membrane microdomains were involved in the localization of GLUTs and uptake of 2-deoxyglucose. We also observed cofractionation of GLUT3 and caveolin-1 in low-buoyant density membranes together with their shift to higher densities after methyl-beta-cyclodextrin treatment. GLUT1 was found in all fractions isolated. Immunofluorescent studies indicated that caveolin-1, GLUT1, and hexokinase I colocalize in spermatocytes while caveolin-1, GLUT3, and hexokinase I colocalize in spermatids. These findings suggest the presence of hexose transporters in DRMDs, and further support a role for intact caveolae or cholesterol-enriched membrane microdomains in relation to glucose uptake and glucose phosphorylation. The results would also explain the different glucose-induced changes in [Ca2+]i in both cells.     

Selective binding of imatinib to the genetic variants of human alpha1-acid glycoprotein

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to alpha1-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of Ka=1.7(+/-0.2)x10(6)M(-1) and n=0.94 could be determined for the binding on the F1-S variant at 37 degrees . Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nKa approximately 3 x 10(4)M(-1)) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.     

Comparative effects of water immersion pretreatment on three different acute pancreatitis models in rats.

Cells respond to stress by upregulating the synthesis of cytoprotective heat shock proteins (HSPs) and antioxidant enzymes. The aim of this study was to compare the effects of cold (CWI) or hot water immersion (HWI) stress on three different acute pancreatitis models (cholecystokinin octapeptide (CCK), sodium taurocholate (TC), and L-arginine (Arg)). We examined the levels of pancreatic HSP60, HSP72, and antioxidants after the water immersion stress. Male Wistar rats were injected with CCK, TC, or Arg at the peak level of pancreatic HSP synthesis, as determined by Western blot analysis. HWI significantly elevated HSP72 expression and CWI significantly increased HSP60 expression in the pancreas. Water immersion stress decreased the levels of pancreatic antioxidants. CWI and-HWI pretreatment ameliorated most of the examined laboratory and morphological parameters of CCK-induced pancreatitis. CWI pretreatment decreased pancreatic edema and the serum amylase level; however, the morphological damage was more severe in TC-induced acute pancreatitis. Overall, CWI and HWI pretreatment only decreased the serum cytokine concentrations in Arg-induced pancreatitis. CWI and HWI resulted in differential induction of pancreatic HSP60 and HSP72 and the depletion of antioxidants. The findings suggest the possible roles of HSP60 and (or) HSP72 (but not that of the antioxidant enzymes) in the protection against CCK- and TC-induced acute pancreatitis. Unexpectedly, CWI pretreatment was detrimental to the morphological parameters of TC-induced pancreatitis. It was demonstrated that CWI and HWI pretreatment only influenced cytokine synthesis in Arg-induced pancreatitis.