Bacterial ATP binding cassette (ABC) transporters are important for the uptake of various nutrients, including essential metals such as zinc. These systems require an extracellular solute binding ...protein (SBP) to bind the substrate with high affinity and specificity and deliver it to the membrane permease for transport. Generally, substrate binding elicits a change from an open to closed conformation that is likely important for permease recognition and transport. However, crystal structures for the metal‐bound and metal‐free forms of several zinc SBPs show relatively subtle or even negligible conformational changes that accompany metal binding. Thus, solution studies are needed for a thorough understanding of the role of conformational dynamics in substrate binding and transport. Here we investigate conformational states and dynamics in a group of zinc SBPs using tryptophan fluorescence intensity and lifetime as a sensitive reporter for the environment of this residue. Useful changes were observed in homologues of the protein AztC from Paracoccus denitrificans and the human pathogen Citrobacter koseri, whereas fluorescence from Citrobacter koseri ZnuA showed no change in fluorescence behavior upon zinc binding. These data combined with isothermal titration calorimetry (ITC) suggest a dynamic equilibrium of at least two conformational states in the apo form and compensatory changes in the holo form that provide for a significant entropic contribution to zinc binding. Correlation with available crystal structures suggests that the formation of a Trp‐Phe pi‐stacking interaction in AztC that is absent in ZnuA may mediate the observed changes in fluorescence. The conformational dynamics identified here highlight diversity in binding mechanisms for zinc SBPs with relevance to the exploitation of these proteins as potential antibiotic drug targets.
Bacteria rely on ATP binding cassette (ABC) transporters for the import of various nutrients. Bacterial ABC importers utilize an extracellular solute binding protein (SBP) to bind the substrate with ...high affinity and specificity and deliver it to the membrane permease for transport. The essential metals iron, manganese, and zinc are bound and transported by the cluster A–I SBPs. Crystal structures exist for the metal-bound and metal-free forms of several cluster A–I SBPs that show relatively subtle conformational changes that accompany metal binding. Recent solution studies and molecular dynamics simulations indicate a more complex conformational landscape for the cluster A–I SBPs, suggesting that changes in protein dynamics upon metal binding may have an important role in recognition by the membrane permease and effective transport. Here, we investigate conformational states and dynamics in the cluster A–I SBP AztC fromParacoccus denitrificans, characterizing its unusual intrinsic fluorescence behavior and thermodynamics of zinc binding. These data suggest a dynamic equilibrium of at least two conformational states in the apo form and compensatory changes in the holo that provide for a significant entropic contribution to zinc binding. Correlation with available crystal structures suggests that the formation of a Trp–Phe π-stacking interaction in the metal-bound form may mediate the observed changes in fluorescence. The conformational dynamics identified here for AztC are likely applicable to other cluster A–I SBPs with relevance to their exploitation as potential antibiotic drug targets.
Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) approved for the treatment of HER2 (human epidermal growth factor receptor 2)-positive breast cancer. T-DM1 consists of trastuzumab ...covalently linked to the cytotoxic maytansinoid DM1 via a non-cleavable linker. Despite its efficacy, primary or acquired resistance frequently develops, particularly in advanced stages of the disease. Second generation ADCs targeting HER2 are meant to supersede T-DM1 by using a cleavable linker and a more potent payload with a different mechanism of action. To determine the effect of one of these novel ADCs, SYD985, on tumors resistant to T-DM1, we developed several patient-derived models of resistance to T-DM1. Characterization of these models showed that previously described mechanisms-HER2 downmodulation, impairment of lysosomal function and upregulation of drug efflux pumps-account for the resistances observed, arguing that mechanisms of resistance to T-DM1 are limited, and most of them have already been described. Importantly, SYD985 was effective in these models, showing that the resistance to first generation ADCs can be overcome with an improved design.
Antibody-drug conjugates (ADC) are antineoplastic agents recently introduced into the antitumor arsenal. T-DM1, a trastuzumab-based ADC that relies on lysosomal processing to release the payload, is ...approved for HER2-positive breast cancer. Next-generation ADCs targeting HER2, such as vic-trastuzumab duocarmazine (SYD985), bear linkers cleavable by lysosomal proteases and membrane-permeable drugs, mediating a bystander effect by which neighboring antigen-negative cells are eliminated. Many antitumor therapies, like DNA-damaging agents or CDK4/6 inhibitors, can induce senescence, a cellular state characterized by stable cell-cycle arrest. Another hallmark of cellular senescence is the enlargement of the lysosomal compartment. Given the relevance of the lysosome to the mechanism of action of ADCs, we hypothesized that therapies that induce senescence would potentiate the efficacy of HER2-targeting ADCs. Treatment with the DNA-damaging agent doxorubicin and CDK4/6 inhibitor induced lysosomal enlargement and senescence in several breast cancer cell lines. While senescence-inducing drugs did not increase the cytotoxic effect of ADCs on target cells, the bystander effect was enhanced when HER2-negative cells were cocultured with HER2-low cells. Knockdown experiments demonstrated the importance of cathepsin B in the enhanced bystander effect, suggesting that cathepsin B mediates linker cleavage. In breast cancer patient-derived xenografts, a combination treatment of CDK4/6 inhibitor and SYD985 showed improved antitumor effects over either treatment alone. These data support the strategy of combining next-generation ADCs targeting HER2 with senescence-inducing therapies for tumors with heterogenous and low HER2 expression.
Combining ADCs against HER2-positive breast cancers with therapies that induce cellular senescence may improve their therapeutic efficacy by facilitating a bystander effect against antigen-negative tumor cells.
The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their ...specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.
Aim: The rich flora of the Caribbean islands and surrounding mainland evolved in a context of isolation alternated with phases of terrestrial connectivity between land-masses, climatic fluctuations ...and episodes of mass extinctions during the Cenozoic. We explored how these events affected the evolution of the sister palm tribes Cryosophileae and Sabaleae, and how continent-island exchanges, endemic radiations and mass extinction shaped their extant diversity. Location: The American continent including the Caribbean region. Methods: We reconstructed a time-calibrated phylogeny of the palm tribes Cryosophileae and Sabaleae using 84% of the known species. We inferred ancestral distribution and tested the effect of island colonization and mass extinction on extant diversity. Results: Our results indicate that Cryosophileae and Sabaleae originated c. 77 Ma most probably in Laurasia, and their extant species started to diversify between 56–35 Ma and 19–10 Ma respectively. Biogeographical state reconstruction estimated that Cryosophileae dispersed to South America between 56–35 Ma, then dispersed to North-Central America between 39–25 Ma and the Caribbean islands between 34–21 Ma. We detected a possible signature of a mass extinction event at the end of the Eocene, affecting the diversification of Cryosophileae and Sabaleae and we did not detect a diversification rate shift related to the colonization of the Caribbean islands. Main conclusions: Species of Cryosophileae in the Caribbean islands are probably derived from a single Oligocene dispersal event that likely occurred Overwater from North-Central America rather than through the hypothesized GAARIandia land bridge. Contrastingly, three independent Miocene dispersal events from North-Central America explain the occurrence of Sabaleae in the Caribbean islands. Contrary to our expectations, island colonization did not trigger increased diversification. Instead, we find that diversification patterns in this clade, and its disappearance from northernmost latitudes, could be the signature of a mass extinction triggered by the global temperature decline at the end of the Eocene.
The winter extratropical teleconnection of El Niño–Southern Oscillation (ENSO) in the North Atlantic–European (NAE) sector remains controversial, concerning both the amplitude of its impacts and the ...underlying dynamics. However, a well-established response is a late-winter (January–March) signal in sea level pressure (SLP) consisting of a dipolar pattern that resembles the North Atlantic Oscillation (NAO). Clarifying the relationship between this “NAO-like” ENSO signal and the actual NAO is the focus of this study. The ENSO–NAE teleconnection and NAO signature are diagnosed by means of linear regression onto the sea surface temperature (SST) Niño-3.4 index and an EOF-based NAO index, respectively, using long-term reanalysis data (NOAA-20CR, ERA-20CR). While the similarity in SLP is evident, the analysis of anomalous upper-tropospheric geopotential height, zonal wind, and transient-eddy momentum flux, as well as precipitation and meridional eddy heat flux, suggests that there is no dynamical link between the phenomena. The observational results are further confirmed by analyzing two 10-member ensembles of atmosphere-only simulations (using an intermediate-complexity and a state-of-the-art model) with prescribed SSTs over the twentieth century. The SST-forced variability in the Northern Hemisphere is dominated by the extratropical ENSO teleconnection, which provides modest but significant SLP skill in the NAE midlatitudes. The regional internally generated variability, estimated from residuals around the ensemble mean, corresponds to the NAO pattern. It is concluded that distinct dynamics are at play in the ENSO–NAE teleconnection and NAO variability, and caution is advised when interpreting the former in terms of the latter.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This work presents observational evidence of a change in Atlantic‐Pacific Niños connection since the late 60's. Accordingly, summer Atlantic Niños (Niñas) alter the tropical circulation favoring the ...development of following‐winter Pacific Niñas (Niños). The same change is obtained in an ensemble of AGCM integrations in which SSTs in the Atlantic are the observed in 1949–2002 and those in the tropical Indo‐Pacific are from a coupled OGCM. The mechanism (for the positive Atlantic phase) involves the strengthening of the Walker circulation with ascending branch over the Atlantic and descending branch over the central Pacific. The enhanced surface divergence in the latter region shallows the equatorial thermocline triggering coupled processes, and favoring the development of a Pacific La Niña. Results could be linked to the reported 60's and 70's climate shifts; emphasizing the importance of tropical Atlantic for the success of seasonal forecast skill.
The Sahel experienced a severe drought during the 1970s and 1980s after wet periods in the 1950s and 1960s. Although rainfall partially recovered since the 1990s, the drought had devastating impacts ...on society. Most studies agree that this dry period resulted primarily from remote effects of sea surface temperature (SST) anomalies amplified by local land surface–atmosphere interactions. This paper reviews advances made during the last decade to better understand the impact of global SST variability on West African rainfall at interannual to decadal time scales. At interannual time scales, a warming of the equatorial Atlantic and Pacific/Indian Oceans results in rainfall reduction over the Sahel, and positive SST anomalies over the Mediterranean Sea tend to be associated with increased rainfall. At decadal time scales, warming over the tropics leads to drought over the Sahel, whereas warming over the North Atlantic promotes increased rainfall. Prediction systems have evolved from seasonal to decadal forecasting. The agreement among future projections has improved from CMIP3 to CMIP5, with a general tendency for slightly wetter conditions over the central part of the Sahel, drier conditions over the western part, and a delay in the monsoon onset. The role of the Indian Ocean, the stationarity of teleconnections, the determination of the leader ocean basin in driving decadal variability, the anthropogenic role, the reduction of the model rainfall spread, and the improvement of some model components are among the most important remaining questions that continue to be the focus of current international projects.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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