Arabidopsis protein S-acyl transferases positively mediate BR signaling through S-acylation of BSK1.

Protein S-acyl transferases (PATs) catalyze S-acylation, a reversible post-translational modification critical for membrane association, trafficking, and stability of substrate proteins. Many plant proteins are potentially S-acylated but few have corresponding PATs identified. By using genomic editing, confocal imaging, pharmacological, genetic, and biochemical assays, we demonstrate that three Arabidopsis class C PATs positively regulate BR signaling through S-acylation of BRASSINOSTEROID-SIGNALING KINASE1 (BSK1). PAT19, PAT20, and PAT22 associate with the plasma membrane (PM) and the trans-Golgi network/early endosome (TGN/EE). Functional loss of all three genes results in a plethora of defects, indicative of reduced BR signaling and rescued by enhanced BR signaling. PAT19, PAT20, and PAT22 interact with BSK1 and are critical for the S-acylation of BSK1, and for BR signaling. The PM abundance of BSK1 was reduced by functional loss of PAT19, PAT20, and PAT22 whereas abolished by its S-acylation-deficient point mutations, suggesting a key role of S-acylation in its PM targeting. Finally, an active BR analog induces vacuolar trafficking and degradation of PAT19, PAT20, or PAT22, suggesting that the S-acylation of BSK1 by the three PATs serves as a negative feedback module in BR signaling.

Mutations in the Drosophila tricellular junction protein M6 synergize with RasV12 to induce apical cell delamination and invasion.

Complications from metastasis are responsible for the majority of cancer-related deaths. Despite the outsized medical impact of metastasis, remarkably little is known about one of the key early steps of metastasis: departure of a tumor cell from its originating tissue. It is well documented that cellular delamination in the basal direction can induce invasive behaviors, but it remains unknown if apical cell delamination can induce migration and invasion in a cancer context. To explore this feature of cancer progression, we performed a genetic screen in Drosophila and discovered that mutations in the protein M6 synergize with oncogenic Ras to drive invasion following apical delamination without crossing a basement membrane. Mechanistically, we observed that M6-deficient RasV12 clones delaminate as a result of alterations in a Canoe-RhoA-myosin II axis that is necessary for both the delamination and invasion phenotypes. To uncover the cellular roles of M6, we show that it localizes to tricellular junctions in epithelial tissues where it is necessary for the structural integrity of multicellular contacts. This work provides evidence that apical delamination can precede invasion and highlights the important role that tricellular junction integrity can play in this process.

Are vacuolar dynamics crucial factors for plant cell division and differentiation?

Vacuoles are the largest membrane-bound organelles in plant cells, critical for development and environmental responses. Vacuolar dynamics indicate reversible changes of vacuoles in morphology, size, or numbers. In this review, we summarize current understandings of vacuolar dynamics in different types of plant cells, biological processes associated with vacuolar dynamics, and regulators controlling vacuolar dynamics. Specifically, we point out the possibility that vacuolar dynamics play key roles in cell division and differentiation, which are controlled by the nucleus. Finally, we propose three routes through which vacuolar dynamics actively participate in nucleus-controlled cellular activities.

S-acylation of YKT61 modulates its unconventional participation in the formation of SNARE complexes in Arabidopsis.

Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells. SNARE assembly involves four different SNARE motifs, Qa, Qb, Qc, and R, provided by three or four SNARE proteins. YKT6 is an atypical R-SNARE that lacks a transmembrane domain and is involved in multiple vesicle-target membrane fusions. Although YKT6 is evolutionarily conserved and essential, its function and regulation in different phyla seem distinct. Arabidopsis YKT61, the yeast and metazoan YKT6 homologue, is essential for gametophytic development, plays a critical role in sporophytic cells, and mediates multiple vesicle-target membrane fusion. However, its molecular regulation is unclear. We report here that YKT61 is S-acylated. Abolishing its S-acylation by a C195S mutation dissociates YKT61 from endomembrane structures and causes its functional loss. Although interacting with various SNARE proteins, YKT61 functions not as a canonical R-SNARE but coordinates with other R-SNAREs to participate in the formation of SNARE complexes. Phylum-specific molecular regulation of YKT6 may be evolved to allow more efficient SNARE assembly in different eukaryotic cells.

The landscape of systemic therapy for early stage triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with higher risk of disease recurrence and mortality than other breast cancer subtypes. Historically, chemotherapy has been the primary systemic treatment for early stage TNBC. Recent developments in immune checkpoint inhibitors (ICIs) and novel therapeutic agents have transformed the treatment of TNBC. AREAS COVERED: This review provides a comprehensive overview of the current evidence on treatment of early stage TNBC. We highlight the incorporation of ICIs and other targeted therapies in (neo)adjuvant treatment and the ongoing development of novel therapeutic agents. EXPERT OPINION: The landscape of early TNBC treatment is rapidly evolving, which has given rise to the introduction of ICIs and PARP inhibitors into the systemic therapy. Despite modest improvement in the pathologic complete response (pCR) rate, ICI plus chemotherapy significantly improves long-term outcomes and is now used in (neo)adjuvant treatment of patients with TNBC and high risk for disease recurrence. Capecitabine remains the standard adjuvant treatment for residual disease, with olaparib being an option for patients with germline BRCA1/2 mutations. Early detection of minimal residual disease may identify patients requiring additional therapy to prevent recurrence.

Hyperpatulones C-G, new spirocyclic polycyclic polyprenylated acylphloroglucinols from the leaves of Hypericum patulum.

Five new spirocyclic polycyclic polyprenylated acylphloroglucinols, Hyperpatulones C-G (1-5), were obtained from the leaves of Hypericum patulum. Their structures were characterized by the comprehensive analysis of their IR, NMR, CD spectra and HRESIMS data. All the new compounds were evaluated for the α-glycosidase inhibitory activities. Among them, compounds 3-5 showed α-glucosidase inhibitory activities, with IC50 values of 14.06-37.69 µM.

Misshapen Disruption Cooperates with RasV12 to Drive Tumorigenesis.

Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. Here, we performed a large-scale genetic screen in Drosophila eye imaginal discs and identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (RasV12) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, we further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, we identified msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, our findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology.

Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance.

Oncogenic RAS mutations are associated with tumor resistance to radiation therapy. Cell-cell interactions in the tumor microenvironment (TME) profoundly influence therapy outcomes. However, the nature of these interactions and their role in Ras tumor radioresistance remain unclear. Here we use Drosophila oncogenic Ras tissues and human Ras cancer cell radiation models to address these questions. We discover that cellular response to genotoxic stress cooperates with oncogenic Ras to activate JAK/STAT non-cell autonomously in the TME. Specifically, p53 is heterogeneously activated in Ras tumor tissues in response to irradiation. This mosaicism allows high p53-expressing Ras clones to stimulate JAK/STAT cytokines, which activate JAK/STAT in the nearby low p53-expressing surviving Ras clones, leading to robust tumor re-establishment. Blocking any part of this cell-cell communication loop re-sensitizes Ras tumor cells to irradiation. These findings suggest that coupling STAT inhibitors to radiotherapy might improve clinical outcomes for Ras cancer patients.

AUF1 is involved in splenic follicular B cell maintenance.

BACKGROUND: The adenosine/uridine-rich element (ARE)-binding protein AUF1 functions to regulate the inflammatory response through the targeted degradation of cytokine and other mRNAs that contain specific AREs in their 3' noncoding region (3' NCR). To investigate the role of AUF1 in the immune system, we characterized the lymphoid compartments of AUF1-deficient mice. RESULTS: Mice lacking AUF1 exhibit an altered proportion and size of splenic B cell subsets. We show prominent apoptosis in splenic B cell follicles and reduced expression of Bcl-2, A1, and Bcl-XL correlate with increased turnover and significant reduction in the number and proportion of splenic FO B cells in AUF1-deficient mice. In addition, AUF1-deficient mice exhibit a sharp decrease in splenic size and lymphocyte cellularity. Bone marrow transfer studies demonstrate that AUF1 deficiency induces cell-autonomous defects in mature B cell subsets but not in the overall number of splenocytes. Reconstitution of irradiated adult AUF1-deficient mice with wild-type bone marrow restores the proportion of FO and marginal zone (MZ) B cells, but does not rescue the decrease in the number of splenocytes. Functionally, AUF1-deficient mice mount an attenuated response to T cell-independent (TI) antigen, which correlates with impaired MZ B cell function. CONCLUSION: These data indicate that AUF1 is important in the maintenance of splenic FO B cells and adequate humoral immune responses.

A novel regulator of ER Ca2+ drives Hippo-mediated tumorigenesis.

Calcium ion (Ca2+) is a versatile second messenger that regulates various cellular and physiological functions. However, the in vivo molecular mechanisms by which Ca2+ alterations contribute to tumor growth remain poorly explored. Here we show that Emei is a novel ER Ca2+ regulator that synergizes with RasV12 to induce tumor growth via JNK-mediated Hippo signaling. Emei disruption reduces ER Ca2+ level and subsequently leads to JNK activation and Hippo inactivation. Importantly, genetically increasing cytosolic Ca2+ concentration cooperates with RasV12 to drive tumor growth via inactivating the Hippo pathway. Finally, we identify POSH as a crucial link that bridges cytosolic Ca2+ alteration with JNK activation and Hippo-mediated tumor growth. Together, our findings provide a novel mechanism of tumor growth that acts through intracellular Ca2+ levels to modulate JNK-mediated Hippo signaling.

PP6 Disruption Synergizes with Oncogenic Ras to Promote JNK-Dependent Tumor Growth and Invasion.

RAS genes are frequently mutated in cancers, yet an effective treatment has not been developed, partly because of an incomplete understanding of signaling within Ras-related tumors. To address this, we performed a genetic screen in Drosophila, aiming to find mutations that cooperate with oncogenic Ras (RasV12) to induce tumor overgrowth and invasion. We identified fiery mountain (fmt), a regulatory subunit of the protein phosphatase 6 (PP6) complex, as a tumor suppressor that synergizes with RasV12 to drive c-Jun N-terminal kinase (JNK)-dependent tumor growth and invasiveness. We show that Fmt negatively regulates JNK upstream of dTAK1. We further demonstrate that disruption of PpV, the catalytic subunit of PP6, mimics fmt loss-of-function-induced tumorigenesis. Finally, Fmt synergizes with PpV to inhibit JNK-dependent tumor progression. Our data here further highlight the power of Drosophila as a model system to unravel molecular mechanisms that may be relevant to human cancer biology.

Clinical manifestations and significance of post-traumatic thoracolumbar syringomyelia.

OBJECTIVE: To analyze the pathogenic mechanism and the clinical significance of post-traumatic thoracolumbar syringomyelia through reviewing the clinical manifestations. METHODS: The data of 15 patients (14 males and 1 female, aged from 28 to 56 years, with an average of 36 years) with post-traumatic syringomyelia treated in our hospital from December 1997 to February 2002 were studied retrospectively. Two patients suffered from T11 fractures, 7 from T12 fractures and 6 from L1 fractures. There were 12 patients with burst fractures and 3 with fracture dislocations. Anterior decompression, bone graft, bone fusion and internal fixation were made on 6 patients, posterior decompression, bone graft, bone fusion and internal fixation on 1 patient, and non-surgical treatment on 8 patients. RESULTS: Syringomyelia of the patients was diagnosed accurately with magnetic resonance imaging at 0.5-4 years after the original thoracolumbar fracture. The cavern was round in 6 cases, elliptic in 6 cases, and irregular in 3 cases. The patients also suffered from pain (80%), myodynamia attenuation in lower extremities (66.7%), aggravated spasm (46.7%), sensation loss or hypesthesia (46.7%), decreased coordinate function of lower extremities (20%) and autonomic nerve symptom (6.7%). CONCLUSIONS: Post-traumatic thoracolumbar syringomyelia should be suspected if the patient has new neurological symptoms, such as myodynamia attenuation in lower extremities, after the neural function becomes stable for certain time.

Endotoxic shock in AUF1 knockout mice mediated by failure to degrade proinflammatory cytokine mRNAs.

Excessive production of proinflammatory cytokines, particularly tumor necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta), plays a critical role in septic shock induced by bacterial endotoxin (endotoxemia). Precise control of cytokine expression depends on rapid degradation of cytokine mRNAs, mediated by an AU-rich element (ARE) in the 3' noncoding region and by interacting ARE-binding proteins, which control the systemic inflammatory response. To understand the function of the ARE-binding protein AUF1, we developed an AUF1 knockout mouse. We show that AUF1 normally functions to protect against the lethal progression of endotoxemia. Upon endotoxin challenge, AUF1 knockout mice display symptoms of severe endotoxic shock, including vascular hemorrhage, intravascular coagulation, and high mortality, resulting from overproduction of TNFalpha and IL-1beta. Overexpression of these two cytokines is specific, and shown to result from an inability to rapidly degrade these mRNAs in macrophages following induction. Neutralizing antibodies to TNFalpha and IL-1beta protect AUF1 knockout mice against lethal endotoxic shock. These and other data describe a novel post-transcriptional mechanism whereby AUF1 acts as a crucial attenuator of the inflammatory response, promoting the rapid decay of selective proinflammatory cytokine mRNAs following endotoxin activation. Defects in the AUF1 post-transcriptionally controlled pathway may be involved in human inflammatory disease.

Assembly of AUF1 with eIF4G-poly(A) binding protein complex suggests a translation function in AU-rich mRNA decay.

An AU-rich element (ARE) located in the 3'-untranslated region of many short-lived mRNAs functions as an instability determinant for these transcripts. AUF1/hnRNP D, an ARE-binding protein family consisting of four isoforms, promotes rapid decay of ARE-mRNAs. The mechanism by which AUF1 promotes rapid decay of ARE-mRNA is unclear. AUF1 has been shown to form an RNase-resistant complex in cells with the cap-initiation complex and heat shock proteins Hsp70 and Hsc70, as well as other unidentified factors. To understand the function of the AUF1 complex, we have biochemically investigated the association of AUF1 with the components of the translation initiation complex. We used purified recombinant proteins and a synthetic ARE RNA oligonucleotide to determine the hierarchy of protein interactions in vitro and the effect of AUF1 binding to the ARE on the formation of protein complexes. We demonstrate that all four AUF1 protein isoforms bind directly and strongly to initiation factor eIF4G at a C-terminal site regardless of AUF1 interaction with the ARE. AUF1 is shown to directly interact with poly(A) binding protein (PABP), both independently of eIF4G and in a complex with eIF4G. AUF1-PABP interaction is opposed by AUF1 binding to the ARE or Hsp70 heat shock protein. In vivo, AUF1 interaction with PABP does not alter PABP stability. Based on these and other data, we propose a model for the molecular interactions of AUF1 that involves translation-dependent displacement of AUF1-PABP complexes from ARE-mRNAs with possible unmasking of the poly(A) tail.

Tissue distribution of AU-rich mRNA-binding proteins involved in regulation of mRNA decay.

Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3'-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function.

Nuclear import and export functions in the different isoforms of the AUF1/heterogeneous nuclear ribonucleoprotein protein family.

The heterogeneous nuclear ribonucleoprotein D family of proteins also known as AUF1 consists of four isoforms implicated in both nuclear and cytoplasmic functions. The AUF1 proteins are largely nuclear but also are found in the cytoplasm and are thought to undergo nucleocytoplasmic shuttling. The nucleocytoplasmic distribution and potential shuttling activity of the individual AUF1 isoforms have not been previously studied in detail. Therefore, we characterized the nucleocytoplasmic transport of each of the heterogeneous nuclear ribonucleoprotein D/AUF1 isoforms. All four AUF1 proteins were found to undergo rapid nucleocytoplasmic shuttling in a manner that is transcription-independent, carrier-mediated, and energy-requiring. Nucleocytoplasmic shuttling of the AUF1 proteins is shown to utilize a novel arrangement of nuclear import and export signals. Mutagenesis of the AUF1 proteins and fusion of polypeptides to a green fluorescent protein reporter demonstrated that a nuclear import signal is located in the C-terminal domain of the protein and is found only in the two smaller isoforms. Further mapping demonstrated that nuclear export is facilitated by sequences in AUF1 exon 7 found in the C-terminal domain of the two larger AUF1 isoforms. A subset of AUF1 proteins are shown to directly interact in vitro using purified recombinant proteins and in vivo in the absence of RNA. These results suggest that nuclear import of AUF1 is facilitated by sequences found only in the two smaller isoforms and that nuclear export is facilitated by sequences (exon 7 and the C-terminal domain) found only in the two larger isoforms. This novel arrangement of signals might represent a mechanism to assure co-shuttling of a subset of AUF1 proteins that interact in a heterocomplex.