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It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging. Trinco et al. measure aspartate uptake rates in proteoliposomes containing purified prokaryotic Na+-coupled aspartate transporter GltTk. To overcome limitation of protein orientation, they use synthetic nanobody that blocks transporters from outside and reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. [ABSTRACT FROM AUTHOR]

This study was aimed at analyzing the relations of metastasis to regional lymph nodes (RLNs) with histopathological indicators of invasive breast carcinoma of no special type (IC-NST) and its cytokine profile. Enzyme-linked immunosorbent assays were performed to determine concentrations of IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1β, IL-1Ra, TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF-A, and MCP-1 in the culture supernatant of IC-NST samples from 48 female patients. Histopathological indicators (degree of tumor cell differentiation, mitoses, and others) and ER, PR, Her2/neu, Ki-67, and CD34 expression levels were determined. By means of three types of neural network models, it was shown that for different parameters of the output layer, different groups of parameters are involved that have predictive value regarding metastasis to RLNs. As a result of multi-dimensional cluster analysis, three clusters were formed with different cytokines profiles of IC-NST. Different correlations between indicators of cytokine production by IC-NST and its histopathological parameters were revealed in groups with different cytokine profiles. It was shown that at simultaneous evaluation of the production of even two cytokines, the importance of which relationship with metastasis was revealed by neural network modeling, can increase the probability of determining the presence of metastasis in the RLNs. [ABSTRACT FROM AUTHOR]

Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue GltTk, a Na+- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na+ ions. These structures explain how substrate-leakage is prevented – a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport. Glutamate transporters are membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft via a so-called elevator mechanism. Here the authors present five cryo-EM structures of the transporter homologue GltTk, which explain how substrate leakage is prevented. [ABSTRACT FROM AUTHOR]

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPFlong. The N-terminal domain of HPFlong binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPFlong binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPFlong, and the Shine-Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPFlong due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPFlong-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria. [ABSTRACT FROM AUTHOR]

Sm and Sm-like proteins represent an evolutionarily conserved family with key roles in RNA metabolism. Sm-based regulation is diverse and can range in scope from eukaryotic mRNA splicing to bacterial quorum sensing, with at least one step in these processes being mediated by an RNA-associated molecular assembly built on Sm proteins. Despite the availability of several 3D-structures of Sm-like archaeal proteins (SmAPs), their function has remained elusive. The aim of this study was to shed light on the function of SmAP1 and SmAP2 of the crenarchaeon Sulfolobus solfataricus (Sso). Using co-purification followed by RNASeq different classes of non-coding RNAs and mRNAs were identified that co-purified either with both paralogues or solely with Sso-SmAP1 or Sso-SmAP2. The large number of associated intron-containing tRNAs and tRNA/rRNA modifying RNAs may suggest a role of the two Sso-SmAPs in tRNA/rRNA processing. Moreover, the 3D structure of Sso-SmAP2 was elucidated. Like Sso-SmAP1, Sso-SmAP2 forms homoheptamers. The binding of both proteins to distinct RNA substrates is discussed in terms of surface conservation, structural differences in the RNA binding sites and differences in the electrostatic surface potential of the two Sso-SmAP proteins. Taken together, this study may hint to common and different functions of both Sso-SmAPs in Sso RNA metabolism. [ABSTRACT FROM AUTHOR]

In eukaryotes and archaea, the heterotrimeric translation initiation factor 2 (e/aIF2) is pivotal for the delivery of methionylated initiator tRNA (Met-tRNAi) to the ribosome. It acts as a molecular switch that cycles between inactive (GDP-bound) and active (GTP-bound) states. Recent studies show that eIF2 can also exist in a long-lived eIF2γ-GDP-Pi (inorganic phosphate) active state. Here, four high-resolution crystal structures of aIF2γ from Sulfolobus solfataricus are reported: aIF2γ-GDPCP (a nonhydrolyzable GTP analogue), aIF2γ-GDP-formate (in which a formate ion possibly mimics Pi), aIF2γ-GDP and nucleotide-free aIF2γ. The structures describe the different states of aIF2γ and demonstrate the conformational transitions that take place in the aIF2γ 'life cycle'. [ABSTRACT FROM AUTHOR]

mRNAs lacking 5′ untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes. [ABSTRACT FROM AUTHOR]