AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells.

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Multimodal cotranslational interactions direct assembly of the human multi-tRNA synthetase complex.

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.

Cotranslational interaction of human EBP50 and ezrin overcomes masked binding site during complex assembly.

Multiprotein assemblages are the intracellular workhorses of many physiological processes. Assembly of constituents into complexes can be driven by stochastic, domain-dependent, posttranslational events in which mature, folded proteins specifically interact. However, inaccessibility of interacting surfaces in mature proteins (e.g., due to "buried" domains) can obstruct complex formation. Mechanisms by which multiprotein complex constituents overcome topological impediments remain enigmatic. For example, the heterodimeric complex formed by EBP50 and ezrin must address this issue as the EBP50-interacting domain in ezrin is obstructed by a self-interaction that occupies the EBP50 binding site. Here, we show that the EBP50-ezrin complex is formed by a cotranslational mechanism in which the C terminus of mature, fully formed EBP50 binds the emerging, ribosome-bound N-terminal FERM domain of ezrin during EZR mRNA translation. Consistent with this observation, a C-terminal EBP50 peptide mimetic reduces the cotranslational interaction and abrogates EBP50-ezrin complex formation. Phosphorylation of EBP50 at Ser339 and Ser340 abrogates the cotranslational interaction and inhibits complex formation. In summary, we show that the function of eukaryotic mRNA translation extends beyond "simple" generation of a linear peptide chain that folds into a tertiary structure, potentially for subsequent complex assembly; importantly, translation can facilitate interactions with sterically inaccessible domains to form functional multiprotein complexes.

Bidirectional Tumor-Promoting Activities of Macrophage Ezrin.

Ezrin links the cytoskeleton to cell surface integrins and plasma membrane receptors, contributing to the proliferative and metastatic potential of cancer cells. Elevated ezrin expression in several cancers is associated with poor outcomes. Tumor cell ezrin expression and function have been investigated in depth; however, its role in macrophages and other tumor microenvironment cells remains unexplored. Macrophages profoundly influence tumorigenesis, and here we explore ezrin's influence on tumor-promoting macrophage functions. Ezrin knockdown in THP-1 macrophages reveals its important contribution to adhesion to endothelial cells. Unexpectedly, ezrin is essential for the basal and breast cancer cell-stimulated THP-1 expression of ITGAM mRNA that encodes integrin CD11b, critical for cell adhesion. Ezrin skews the differentiation of THP-1 macrophages towards the pro-tumorigenic, M2 subtype, as shown by the reduced expression of FN1, IL10, and CCL22 mRNAs following ezrin knockdown. Additionally, macrophage ezrin contributes to the secretion of factors that stimulate tumor cell migration, invasion, and clonogenic growth. Lastly, THP-1 ezrin is critical for the expression of mRNAs encoding vascular endothelial growth factor (VEGF)-A and matrix metalloproteinase (MMP)-9, consistent with pro-tumorigenic function. Collectively, our results provide insight into ezrin's role in tumorigenesis, revealing a bidirectional interaction between tumor-associated macrophages and tumor cells, and suggest myeloid cell ezrin as a target for therapeutic intervention against cancer.

Functions for rice RFL in vegetative axillary meristem specification and outgrowth.

Axillary meristems (AMs) are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers, and tillering mutants reveal an interplay between transcription factors and the phytohormones auxin and strigolactone as some factors that underpin this developmental process. Previous studies showed that knockdown of the transcription factor gene RFL reduced tillering and caused a very large decrease in panicle branching. Here, the relationship between RFL, AM initiation, and outgrowth was examined. We show that RFL promotes AM specification through its effects on LAX1 and CUC genes, as their expression was modulated on RFL knockdown, on induction of RFL:GR fusion protein, and by a repressive RFL-EAR fusion protein. Further, we report reduced expression of auxin transporter genes OsPIN1 and OsPIN3 in the culm of RFL knockdown transgenic plants. Additionally, subtle change in the spatial pattern of IR4 DR5:GFP auxin reporter was observed, which hints at compromised auxin transport on RFL knockdown. The relationship between RFL, strigolactone signalling, and bud outgrowth was studied by transcript analyses and by the tillering phenotype of transgenic plants knocked down for both RFL and D3. These data suggest indirect RFL-strigolactone links that may affect tillering. Further, we show expression modulation of the auxin transporter gene OsPIN3 upon RFL:GR protein induction and by the repressive RFL-EAR protein. These modified forms of RFL had only indirect effects on OsPIN1. Together, we have found that RFL regulates the LAX1 and CUC genes during AM specification, and positively influences the outgrowth of AMs though its effects on auxin transport.

Myeloid-cell-specific role of Gasdermin D in promoting lung cancer progression in mice.

The activities of the NLRP3 and AIM2 inflammasomes and Gasdermin D (GsdmD) are implicated in lung cancer pathophysiology but it's not clear if their contributions promote or retard lung cancer progression. Using a metastatic Lewis lung carcinoma (LLC) cell model, we show that GsdmD knockout (GsdmD-/-) mice form significantly fewer cancer foci in lungs, exhibit markedly decreased lung cancer metastasis, and show a significant ∼50% increase in median survival rate. The cleaved forms of GsdmD and IL-1ß were detected in lung tumor tissue, indicating inflammasome activity in lung tumor microenvironment (TME). Increased migration and growth of LLC cells was observed upon exposure to the conditioned media derived from inflammasome-induced wild type, but not the GsdmD-/-, macrophages. Using bone marrow transplantations, we show a myeloid-specific contribution of GsdmD in lung cancer metastasis. Taken together, our data show that GsdmD plays a myeloid-specific role in lung cancer progression.

Rigor, Reproducibility, and Transparency in Shared Research Resources: Follow-Up Survey and Recommendations for Improvements.

Rigor, reproducibility, and transparency (RR&T) are essential components of all scientific pursuits. Shared research resources, also known as core facilities, are on the frontlines of ensuring robust RR&T practices. The Association of Biomolecular Resource Facilities Committee on Core Rigor and Reproducibility conducted a follow-up survey 4 years after the initial 2017 survey to determine if core facilities have seen a positive impact of new RR&T initiatives (including guidance from the National Institutes of Health, new scientific journal requirements on transparency and data provenance, and educational tools from professional organizations). While there were fewer participants in the most recent survey, the respondents' opinions on the role of core facilities and level of best practices adoption remained the same. Overall, the respondents agreed that procedures should be implemented by core facilities to ensure scientific RR&T. They also indicated that there is a strong correlation between institutions that emphasize RR&T and core customers using this expertise in grant applications and publications. The survey also assessed the impact of the COVID-19 pandemic on core operations and RR&T. The answers to these pandemic-related questions revealed that many of the strategies aimed at increasing efficiencies are also best practices related to RR&T, including the development of standard operating procedures, supply chain management, and cross training. Given the consistent and compelling awareness of the importance of RR&T expressed by core directors in 2017 and 2021 contrasted with the lack of apparent improvements over this time period, the authors recommend an adoption of RR&T statements by all core laboratories. Adhering to the RR&T guidelines will result in more efficient training, better compliance, and improved experimental approaches empowering cores to become "rigor champions."

Organic solvent tolerance of Halobacterium sp. SP1(1) and its extracellular protease.

Halophilic archaea belonging to three different genera- Halobacterium, Haloarcula and Haloferax, were isolated from Kandla salt pans. The isolates had an optimum requirement of 25% NaCl for growth. Increase in organic solvent tolerance of isolates was observed at higher NaCl concentrations. Among the three isolates Halobacterium sp. SP1(1) was found to be more tolerant than Haloarcula sp. SP2(2) and Haloferax sp. SP1(2a). The extracellular protease of Halobacterium sp. SP1(1) showed higher solvent tolerance compared to the organism itself. The enzyme was highly tolerant to toluene, xylene, n-decane, n-dodecane and n-undecane, majority of which are frequently used in paints. These findings may help in understanding the mechanism of organic solvent tolerance in halophilic archaea and their application in antifouling coatings. Also, best to our knowledge the present study is the first report on organic solvent tolerance of haloarchaeal extracellular protease.