Partial cooperative unfolding in proteins as observed by hydrogen exchange mass spectrometry

This is an Accepted Manuscript of an article published by Taylor & Francis in International Reviews in Physical Chemistry on 2013-1-1, available online: http://www.tandfonline.com/10.1080/0144235X.2012.751175.Many proteins do not exist in a single rigid conformation. Protein motions, or dynamics, exist and in many cases are important for protein function. The analysis of protein dynamics relies on biophysical techniques that can distinguish simultaneously existing populations of molecules and their rates of interconversion. Hydrogen exchange (HX) detected by mass spectrometry (MS) is contributing to our understanding of protein motions by revealing unfolding and dynamics on a wide timescale, ranging from seconds to hours to days. In this review we discuss HX MS-based analyses of protein dynamics, using our studies of multi-domain kinases as examples. Using HX MS, we have successfully probed protein dynamics and unfolding in the isolated SH3, SH2 and kinase domains of the c-Src and Abl kinase families, as well as the role of inter- and intra-molecular interactions in the global control of kinase function. Coupled with high-resolution structural information, HX MS has proved to be a powerful and versatile tool for the analysis of the conformational dynamics in these kinase systems, and has provided fresh insight regarding the regulatory control of these important signaling proteins. HX MS studies of dynamics are applicable not only to the proteins we illustrate here, but to a very wide range of proteins and protein systems, and should play a role in both classification of and greater understanding of the prevalence of protein motion

mRNAs containing the histone 3' stem-loop are degraded primarily by decapping mediated by oligouridylation of the 3' end

Metazoan replication-dependent histone mRNAs are only present in S-phase, due partly to changes in their stability. These mRNAs end in a unique stem–loop (SL) that is required for both translation and cell-cycle regulation. Previous studies showed that histone mRNA degradation occurs through both 5′→3′ and 3′→5′ processes, but the relative contributions are not known. The 3′ end of histone mRNA is oligouridylated during its degradation, although it is not known whether this is an essential step. We introduced firefly luciferase reporter mRNAs containing the histone 3′ UTR SL (Luc-SL) and either a normal or hDcp2-resistant cap into S-phase HeLa cells. Both mRNAs were translated, and translation initially protected the mRNAs from degradation, but there was a lag of ∼40 min with the uncleavable cap compared to ∼8 min for the normal cap before rapid decay. Knockdown of hDcp2 resulted in a similar longer lag for Luc-SL containing a normal cap, indicating that 5′→3′ decay is important in this system. Inhibition of DNA replication with hydroxyurea accelerated the degradation of Luc-SL. Knockdown of terminal uridyltransferase (TUTase) 4 but not TUTase 3 slowed the decay process, but TUTase 4 knockdown had no effect on destabilization of the mRNA by hydroxyurea. Both Luc-SL and its 5′ decay intermediates were oligouridylated. Preventing oligouridylation by 3′-deoxyadenosine (cordycepin) addition to the mRNA slowed degradation, in the presence or absence of hydroxyurea, suggesting oligouridylation initiates degradation. The spectrum of oligouridylated fragments suggests the 3′→5′ degradation machinery stalls during initial degradation, whereupon reuridylation occurs

Life span and reproductive cost explain interspecific variation in the optimal onset of reproduction.

Fitness can be profoundly influenced by the age at first reproduction (AFR), but to date the AFR-fitness relationship only has been investigated intraspecifically. Here, we investigated the relationship between AFR and average lifetime reproductive success (LRS) across 34 bird species. We assessed differences in the deviation of the Optimal AFR (i.e., the species-specific AFR associated with the highest LRS) from the age at sexual maturity, considering potential effects of life history as well as social and ecological factors. Most individuals adopted the species-specific Optimal AFR and both the mean and Optimal AFR of species correlated positively with life span. Interspecific deviations of the Optimal AFR were associated with indices reflecting a change in LRS or survival as a function of AFR: a delayed AFR was beneficial in species where early AFR was associated with a decrease in subsequent survival or reproductive output. Overall, our results suggest that a delayed onset of reproduction beyond maturity is an optimal strategy explained by a long life span and costs of early reproduction. By providing the first empirical confirmations of key predictions of life-history theory across species, this study contributes to a better understanding of life-history evolution

Winter Bird Assemblages in Rural and Urban Environments: A National Survey

Urban development has a marked effect on the ecological and behavioural traits of many living organisms, including birds. In this paper, we analysed differences in the numbers of wintering birds between rural and urban areas in Poland. We also analysed species richness and abundance in relation to longitude, latitude, human population size, and landscape structure. All these parameters were analysed using modern statistical techniques incorporating species detectability. We counted birds in 156 squares (0.25 km2 each) in December 2012 and again in January 2013 in locations in and around 26 urban areas across Poland (in each urban area we surveyed 3 squares and 3 squares in nearby rural areas). The influence of twelve potential environmental variables on species abundance and richness was assessed with Generalized Linear Mixed Models, Principal Components and Detrended Correspondence Analyses. Totals of 72 bird species and 89,710 individual birds were recorded in this study. On average (±SE) 13.3 ± 0.3 species and 288 ± 14 individuals were recorded in each square in each survey. A formal comparison of rural and urban areas revealed that 27 species had a significant preference; 17 to rural areas and 10 to urban areas. Moreover, overall abundance in urban areas was more than double that of rural areas. There was almost a complete separation of rural and urban bird communities. Significantly more birds and more bird species were recorded in January compared to December. We conclude that differences between rural and urban areas in terms of winter conditions and the availability of resources are reflected in different bird communities in the two environments

Cyclic Expression of Lhx2 Regulates Hair Formation

Hair is important for thermoregulation, physical protection, sensory activity, seasonal camouflage, and social interactions. Hair is generated in hair follicles (HFs) and, following morphogenesis, HFs undergo cyclic phases of active growth (anagen), regression (catagen), and inactivity (telogen) throughout life. The transcriptional regulation of this process is not well understood. We show that the transcription factor Lhx2 is expressed in cells of the outer root sheath and a subpopulation of matrix cells during both morphogenesis and anagen. As the HFs enter telogen, expression becomes undetectable and reappears prior to initiation of anagen in the secondary hair germ. In contrast to previously published results, we find that Lhx2 is primarily expressed by precursor cells outside of the bulge region where the HF stem cells are located. This developmental, stage- and cell-specific expression suggests that Lhx2 regulates the generation and regeneration of hair. In support of this hypothesis, we show that Lhx2 is required for anagen progression and HF morphogenesis. Moreover, transgenic expression of Lhx2 in postnatal HFs is sufficient to induce anagen. Thus, our results reveal an alternative interpretation of Lhx2 function in HFs compared to previously published results, since Lhx2 is periodically expressed, primarily in precursor cells distinct from those in the bulge region, and is an essential positive regulator of hair formation

Does the Order of Invasive Species Removal Matter? The Case of the Eagle and the Pig

Invasive species are recognized as a primary driver of native species endangerment and their removal is often a key component of a conservation strategy. Removing invasive species is not always a straightforward task, however, especially when they interact with other species in complex ways to negatively influence native species. Because unintended consequences may arise if all invasive species cannot be removed simultaneously, the order of their removal is of paramount importance to ecological restoration. In the mid-1990s, three subspecies of the island fox Urocyon littoralis were driven to near extinction on the northern California Channel Islands owing to heightened predation by golden eagles Aquila chrysaetos. Eagles were lured to the islands by an abundant supply of feral pigs Sus scrofa and through the process of apparent competition pigs indirectly facilitated the decline in foxes. As a consequence, both pigs and eagles had to be removed to recover the critically endangered fox. Complete removal of pigs was problematic: removing pigs first could force eagles to concentrate on the remaining foxes, increasing their probability of extinction. Removing eagles first was difficult: eagles are not easily captured and lethal removal was politically distasteful.Using prey remains collected from eagle nests both before and after the eradication of pigs, we show that one pair of eagles that eluded capture did indeed focus more on foxes. These results support the premise that if the threat of eagle predation had not been mitigated prior to pig removal, fox extinction would have been a more likely outcome.If complete eradication of all interacting invasive species is not possible, the order in which they are removed requires careful consideration. If overlooked, unexpected consequences may result that could impede restoration

Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process

A unified phylogeny-based nomenclature for histone variants

Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure