Identification of two imidazole binding sites and key residues for substrate specificity in human primary amine oxidase AOC3

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 ? sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 ? structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.     

Approaches for inclusion of forest carbon cycle in life cycle assessment – a review

Forests are a significant pool of terrestrial carbon. A key feature related to forest biomass harvesting and use is the typical time difference between carbon release into and sequestration from the atmosphere. Traditionally, the use of sustainably grown biomass has been considered as carbon neutral in life cycle assessment (LCA) studies. However, various approaches to account for greenhouse gas (GHG) emissions and sinks of forest biomass acquisition and use have also been developed and applied, resulting in different conclusions on climate impacts of forest products. The aim of this study is to summarize, clarify, and assess the suitability of these approaches for LCA. A literature review is carried out, and the results are analyzed through an assessment framework. The different approaches are reviewed through their approach to the definition of reference land‐use situation, consideration of time frame and timing of carbon emissions and sequestration, substitution credits, and indicators applied to measure climate impacts. On the basis of the review, it is concluded that, to account for GHG emissions and the related climate impacts objectively, biomass carbon stored in the products and the timing of sinks and emissions should be taken into account in LCA. The reference situation for forest land use has to be defined appropriately, describing the development in the absence of the studied system. We suggest the use of some climate impact indicator that takes the timing of the emissions and sinks into consideration and enables the use of different time frames. If substitution credits are considered, they need to be transparently presented in the results. Instead of carbon stock values taken from the literature, the use of dynamic forest models is recommended.     

Dual role of FoxA1 in androgen receptor binding to chromatin, androgen signalling and prostate cancer

High androgen receptor (AR) level in primary tumour predicts increased prostate cancer-specific mortality. However, the mechanisms that regulate AR function in prostate cancer are poorly known. We report here a new paradigm for the forkhead protein FoxA1 action in androgen signalling. Besides pioneering the AR pathway, FoxA1 depletion elicited extensive redistribution of AR-binding sites (ARBs) on LNCaP-1F5 cell chromatin that was commensurate with changes in androgen-dependent gene expression signature. We identified three distinct classes of ARBs and androgen-responsive genes: (i) independent of FoxA1, (ii) pioneered by FoxA1 and (iii) masked by FoxA1 and functional upon FoxA1 depletion. FoxA1 depletion also reprogrammed AR binding in VCaP cells, and glucocorticoid receptor binding and glucocorticoid-dependent signalling in LNCaP-1F5 cells. Importantly, FoxA1 protein level in primary prostate tumour had significant association to disease outcome; high FoxA1 level was associated with poor prognosis, whereas low FoxA1 level, even in the presence of high AR expression, predicted good prognosis. The role of FoxA1 in androgen signalling and prostate cancer is distinctly different from that in oestrogen signalling and breast cancer.     

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A

We have analyzed the structure and function of the integrin α₁I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α₁I C139S/E317A was a higher avidity collagen binder than α₁I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α₁I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α₂I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α₂I structure, has not changed its position in the activated α₁I variant. During the integrin activation, Glu³³⁵ on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β₁ subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu³³⁵. This indicates that the stabilization of helix 7 into its downward position is not required if the α₁ MIDAS is already open. To conclude, the activated α₁I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg²⁸⁷-Glu³¹⁷ ion pair has just broken during the integrin activation.     

Zooplankton as indicators in lakes: a scientific-based plea for including zooplankton in the ecological quality assessment of lakes according to the European Water Framework Directive (WFD)

With the implementation of the EU Water Framework Directive (WFD), the member states have to classify the ecological status of surface waters following standardised procedures. It was a matter of some surprise to lake ecologists that zooplankton were not included as a biological quality element (BQE) despite their being considered to be an important and integrated component of the pelagic food web. To the best of our knowledge, the decision of omitting zooplankton is not wise, and it has resulted in the withdrawal of zooplankton from many so-far-solid monitoring programmes. Using examples from particularly Danish, Estonian, and the UK lakes, we show that zooplankton (sampled from the water and the sediment) have a strong indicator value, which cannot be covered by sampling fish and phytoplankton without a very comprehensive and costly effort. When selecting the right metrics, zooplankton are cost-efficient indicators of the trophic state and ecological quality of lakes. Moreover, they are important indicators of the success/failure of measures taken to bring the lakes to at least good ecological status. Therefore, we strongly recommend the EU to include zooplankton as a central BQE in the WFD assessments, and undertake similar regional calibration exercises to obtain relevant and robust metrics also for zooplankton as is being done at present in the cases of fish, phytoplankton, macrophytes and benthic invertebrates.     

Morphometry and trophic state modify the thermal response of lakes to meteorological forcing

We hypothesised that (i) a summer heat wave would increase the thermal stability of lakes and (ii) the size and trophic state differences would modify the lakes’ responses to heating. Within 2 years, 2008 and 2009, we studied the thermal and optical regimes of two adjacent stratified lakes in northern Italy, the oligo-mesotrophic Lake Monate (2.5 km², max. depth 34 m) and the eutrophic Lake Varese (14.8 km², 26 m). After the cold winter 2008–2009, a heat wave starting in May turned the whole year 2009 the second hottest after 2003. The particular sequence of meteorological events resulted in extreme vertical temperature gradients and unusually high thermal stability of both lakes. All calculated thermal parameters showed the highest values in 2009 while also the values for 2008 exceeded considerably those published for these lakes in the past. Due to the large wind exposed surface, wind mixing was supposedly the dominating mechanism of heat transfer in the shallower eutrophic Lake Varese where, due to low water transparency, large amount of solar energy trapped in the upper layers markedly increased the thermal stability. In the deeper and more transparent Lake Monate, the deeper penetrating solar irradiance contributed to better energy dissipation within the water column and smaller interannual differences in thermal stability.     

Phosphorus retention as a function of external loading,hydraulic turnover time,area and relative depth in 54 lakes and reservoirs

We analysed phosphorus retention as a function of external loading, hydraulic turnover time, area and relative depth on the basis of published data from 54 lakes and reservoirs in different climate regions around the world. Our analysis demonstrated that reservoirs and lakes that received higher areal loading of phosphorus (TP_in) also retained more P per m² but the proportion of the external P loading retained in the waterbody (retention coefficient, R _P) remained generally independent of TP_in. The waterbodies with longer hydraulic residence times (T _R) retained larger proportions of external P and the correlation between R _P and T _R was much stronger in lakes with areas larger than 25 km² than in the whole data set. TP_in and T _R together determined 78% of the variation in R _P in large lakes. We also partially confirmed our hypothesis that waterbodies with bigger relative depths (Z _R) retain more of the external phosphorus than larger and shallower waterbodies with lower Z _R. The hypothesis was, however, validated only for lakes larger than 25 km² and for those with T _R <0.3 year, where R _P increased significantly with increasing Z _R. In stratified lakes, increasing relative depth correlated with reduced P retention capacity, demonstrating the complex nature of phosphorus biogeochemistry in lake ecosystems.     

Transformation system for Hypocrea jecorina (Trichoderma reesei) that favors homologous integration and employs reusable bidirectionally selectable markers

Hypocrea jecorina is an industrially important filamentous fungus due to its effective production of hydrolytic enzymes. It has received increasing interest because of its ability to convert lignocellulosic biomass to monomeric sugars, which can be converted into biofuels or platform chemicals. Genetic engineering of strains is a highly important means of meeting the requirements of tailor-made applications. Therefore, we report the development of a transformation system that allows highly efficient gene targeting by using a tmus53 (human LIG4 homolog) deletion strain. Moreover, it permits the unlimited reuse of the same marker by employing a Cre/loxP-based excision system. Both marker insertion and marker excision can be positively selected for by combining resistance to hygromycin B and loss of sensitivity to fluoroacetamide. Finally, the marker pyr4, also positively selectable for insertion and loss, can be used to remove the cre gene.