Comparative analysis of the Band 4.1/ezrin-related protein tyrosine phosphatase Pez from two Drosophila species: implications for structure and function

The FERM-PTPs are a group of proteins that have FERM (Band 4.1, ezrin, radixin, moesin homology) domains at or near their N-termini, and PTP (protein tyrosine phosphatase) domains at their C-termini. Their central regions contain either PSD-95, Dlg, ZO-1 homology domains or putative Src homology 3 domain binding sites. The known FERM-PTPs fall into three distinct classes, which we name BAS, MEG, and PEZ, after representative human PTPs. Here we analyze Pez, a novel gene encoding the single PEZ-class protein present in Drosophila. Pez cDNAs were sequenced from the distantly related flies Drosophila melanogaster and Drosophila silvestris, and found to be highly conserved except in the central region, which contains at least 21 insertions and deletions. Comparison of fly and human Pez reveals several short conserved motifs in the central region that are likely protein binding sites and/or phosphorylation sites. We also identified novel invertebrate members of the BAS and MEG classes using genome data, and generated an alignment of vertebrate and invertebrate FERM domains of each class. ‘Specialized’ residues were identified that are conserved only within a given class of PTPs. These residues highlight surface regions that may bind class-specific ligands; for PEZ, these residues cluster on and near FERM subdomain F1. Finally, the PTP domain of fly Pez was modeled based on known PTP tertiary structures, and we conclude that Pez is likely a functional phosphatase despite some unusual features of the active site cleft sequences. Biochemical confirmation of this hypothesis and genetic analysis of Pez are currently underway

Post-fire Management Affects Species Composition but not Douglas-fir Regeneration in the Klamath Mountains

Ensuring adequate conifer regeneration after high severity wildfires is a common objective for ecologists and forest managers. In the Klamath region of Oregon and California, a global hotspot of botanical biodiversity, concerns over regeneration have led to post-fire management on many sites, which involves salvage logging followed by site preparation, conifer planting, and manual shrub release. To quantify the impacts of post-fire management, we sampled 62 field sites that burned at high severity nearly 20 years ago in the Klamath-Siskiyou Mountain bioregion, stratifying by management and aspect. We measured cover of shrubs and trees and density and frequency of trees and used Nonmetric Multidimensional Scaling to compare community composition, plant community assemblage based on regenerative traits, and density of tree species between aspect and management. On average, shrub cover exceeded the cover of conifers, hardwoods or grasses, regardless of management history or aspect. The average number of species was lower and resprouting species were less abundant on south aspect sites; seed banking species were most abundant on north aspects. Post-fire management was associated with greater cover of seed banking and nitrogen-fixing species but it did not affect diversity. Management had no impact on Douglas-fir regeneration, the main species of concern in the region. Regeneration of ponderosa pine was higher on sites with post-fire management, but only on south slopes. The frequency of Douglas-fir was associated with aspect, while the frequency of ponderosa pine was associated with management. Overall, our study demonstrates the important role that aspect plays in determining the effectiveness of management after high severity wildfires. Indeed, the effect of aspect on site conditions often overwhelmed the ability of management to influence community composition (including different regenerative strategies), species diversity, and regeneration. Managed sites differed from unmanaged sites in several diverse ways with varied implications for longer-term forest development. Managed sites had taller dominant conifers, which suggests that post-fire management could hasten the period needed to achieve fire resistance. Managed sites were similar in plant community composition but had more homogeneous structure--e.g., managed sites had fewer snags, which are an important habitat feature for several bird species in the region. Finally, management was not associated with conifer regeneration success on north slopes, suggesting that interventions may not be needed uniformly across post-fire landscape. New policies of ecosystem-based management in the Klamath region should consider the important interactions between aspect and post-fire management, and tailor management practices based on specific objectives and landscape context

Thermodynamic consequences of Tyr to Trp mutations in the cation-π-mediated binding of trimethyllysine by the HP1 chromodomain.

Evolution has converged on cation-π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-π interaction is about -5 kcal mol-1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples