TFPIα anticoagulant function is highly dependent on protein S in vivo.

Tissue factor pathway inhibitor α (TFPIα) is the major physiological regulator of the initiation of blood coagulation. In vitro, TFPIα anticoagulant function is enhanced by its cofactor, protein S. To define the role of protein S enhancement in TFPIα anticoagulant function in vivo, we blocked endogenous TFPI in mice using a monoclonal antibody (14D1). This caused a profound increase in fibrin deposition using the laser injury thrombosis model. To explore the role of plasma TFPIα in regulating thrombus formation, increasing concentrations of human TFPIα were coinjected with 14D1, which dose-dependently reduced fibrin deposition. Inhibition of protein S cofactor function using recombinant C4b-binding protein ß chain significantly reduced the anticoagulant function of human TFPIα in controlling fibrin deposition. We report an in vivo model that is sensitive to the anticoagulant properties of the TFPIα-protein S pathway and show the importance of protein S as a cofactor in the anticoagulant function of TFPIα in vivo.

Safe and Efficacious Near Superhydrophobic Hemostat for Reduced Blood Loss and Easy Detachment in Traumatic Wounds.

Hemorrhage is the leading cause of trauma death, and innovation in hemostatic technology is important. The strongly hydrophobic carbon nanofiber (CNF) coating has previously been shown to have excellent hemostatic properties. However, the understanding of how CNF coating guides the coagulation cascade and the biosafety of CNF as hemostatic agents has yet to be explored. Here, our thrombin generation assay investigation showed that CNF induced fast blood coagulation via factor (F) XII activation of the intrinsic pathway. We further performed studies of a rat vein injury and demonstrated that the CNF gauze enabled a substantial reduction of blood loss compared to both the plain gauze and kaolin-imbued gauze (QuikClot). Analysis of blood samples from the model revealed no acute toxicity from the CNF gauze, with no detectable CNF deposition in any organ, suggesting that the immobilization of CNF on our gauze prevented the infiltration of CNF into the bloodstream. Direct injection of CNF into the rat vein was also investigated and found not to elicit overt acute toxicity or affect animal survival or behavior. Finally, toxicity assays with primary keratinocytes revealed minimal toxicity responses to CNF. Our studies thus supported the safety and efficacy of the CNF hemostatic gauze, highlighting its potential as a promising approach in the field of hemostatic control.

Adult human kidney organoids originate from CD24+ cells and represent an advanced model for adult polycystic kidney disease.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24+ epithelial subpopulation. Long-term-cultured CD24+ cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR-Cas9 gene-edited PKD1- and PKD2-knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling.

Spatial multi-omic map of human myocardial infarction.

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.

Decoding myofibroblast origins in human kidney fibrosis.

Kidney fibrosis is the hallmark of chronic kidney disease progression; however, at present no antifibrotic therapies exist1-3. The origin, functional heterogeneity and regulation of scar-forming cells that occur during human kidney fibrosis remain poorly understood1,2,4. Here, using single-cell RNA sequencing, we profiled the transcriptomes of cells from the proximal and non-proximal tubules of healthy and fibrotic human kidneys to map the entire human kidney. This analysis enabled us to map all matrix-producing cells at high resolution, and to identify distinct subpopulations of pericytes and fibroblasts as the main cellular sources of scar-forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single-cell RNA sequencing and ATAC-seq (assay for transposase-accessible chromatin using sequencing) experiments in mice, and spatial transcriptomics in human kidney fibrosis, to shed light on the cellular origins and differentiation of human kidney myofibroblasts and their precursors at high resolution. Finally, we used this strategy to detect potential therapeutic targets, and identified NKD2 as a myofibroblast-specific target in human kidney fibrosis.

Crystal structure and substrate-induced activation of ADAMTS13.

Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.

Differential response to ablative ionizing radiation in genetically distinct non-small cell lung cancer cells.

Stereotactic ablative radiotherapy (SABR) has emerged as a highly promising treatment for medically inoperable early-stage non-small cell lung cancer patients. Treatment outcomes after SABR have been excellent compared to conventional fractionated radiotherapy (CFRT). However, the biological determinants of the response to ablative doses of radiation remain poorly characterized. Furthermore, there's little data on the cellular and molecular response of genetically distinct NSCLC subtypes to radiation. We assessed the response of 3 genetically distinct lung adenocarcinoma cell lines to ablative and fractionated ionizing radiation (AIR and FIR). We studied clonogenic survival, cell proliferation, migration, invasion, apoptosis and senescence. We also investigated the effect of AIR and FIR on the expression of pro-invasive proteins, epithelial-to-mesenchymal transition (EMT), extracellular signal-regulated kinases (ERK1/2) and the transmembrane receptor cMET. Our findings reveal that AIR significantly reduced cell proliferation and clonogenic survival compared to FIR in A549 cells only. This differential response was not observed in HCC827 or H1975 cells. AIR significantly enhanced the invasiveness of A549 cells, but not HCC827 or H1975 cells compared to FIR. Molecular analysis of pathways involved in cell proliferation and invasion revealed that AIR significantly reduced phosphorylation of ERK1/2 and upregulated cMET expression in A549 cells. Our results show a differential proliferative and invasive response to AIR that is dependent on genetic subtype and independent of intrinsic radioresistance. Further examination of these findings in a larger panel of NSCLC cell lines and in pre-clinical models is warranted for identification of biomarkers of tumor response to AIR.

[Raman Spectra Study on the Structure and Thermal Stability of Vacuum Annealed Cr-DLC Films Prepared with UBMS].

Chromium doped amorphous hydrogenated carbon a-C∶H(Cr) films (2.23 µm in thickness)have been successfully prepared with unbalanced magnetron sputtering technology(UBMS) in this paper. Raman spectrum and X-ray photoelectron spectroscopy (XPS) have been used to study the structure and thermal stability of the Chromium doped diamond-like carbon films (Cr-DLC). The results indicate that, at the room temperature, the Raman absorption peak "G" near 1 580 cm-1was assigned to C­C stretching vibration mode (E2g) in the Cr-DLC films, while the peak "D" near 1 350 cm-1was assigned to sp2 breathing vibration mode (A1g). The relative concentration of sp3 bond in the film is at 48%. When heated till 300 ℃, the Raman pattern of sample is similar to that of the sample at room temperature, indicating the structure of Cr-DLC film is stable. At 400 ℃, the ratio of ID/IG and the concentration of sp2 bond clearly increase, indicating the change of the structure in the Cr-DLC and the appearance of graphitization. With temperature going up, the rise of ID/IG ratio and the shift of the peak "G" to high wavenumber show that the rise of sp2/sp3 ratio, the intensity of graphitization, and the decline of the disorder of sp2 in the Cr-DLC films lead to the increase of the friction coefficient and wearing rate and the decline of the Cr-DLC thermal stability. When the it is up to 600 ℃, the ratio of ID/IG and the concentration of sp2 bond reach the max value while the Cr-DLC film loses its efficacy.

The Polycystin-1, Lipoxygenase, and α-Toxin Domain Regulates Polycystin-1 Trafficking.

Mutations in polycystin-1 (PC1) give rise to autosomal dominant polycystic kidney disease, an important and common cause of kidney failure. Despite its medical importance, the function of PC1 remains poorly understood. Here, we investigated the role of the intracellular polycystin-1, lipoxygenase, and α-toxin (PLAT) signature domain of PC1 using nuclear magnetic resonance, biochemical, cellular, and in vivo functional approaches. We found that the PLAT domain targets PC1 to the plasma membrane in polarized epithelial cells by a mechanism involving the selective binding of the PLAT domain to phosphatidylserine and L-α-phosphatidylinositol-4-phosphate (PI4P) enriched in the plasma membrane. This process is regulated by protein kinase A phosphorylation of the PLAT domain, which reduces PI4P binding and recruits ß-arrestins and the clathrin adaptor AP2 to trigger PC1 internalization. Our results reveal a physiological role for the PC1-PLAT domain in renal epithelial cells and suggest that phosphorylation-dependent internalization of PC1 is closely linked to its function in renal development and homeostasis.

Elevated ARG1 expression in primary monocytes-derived macrophages as a predictor of radiation-induced acute skin toxicities in early breast cancer patients.

Radiation therapy (RT) the front-line treatment after surgery for early breast cancer patients is associated with acute skin toxicities in at least 40% of treated patients. Monocyte-derived macrophages are polarized into functionally distinct (M1 or M2) activated phenotypes at injury sites by specific systemic cytokines known to play a key role in the transition between damage and repair in irradiated tissues. The role of M1 and M2 macrophages in RT-induced acute skin toxicities remains to be defined. We investigated the potential value of M1 and M2 macrophages as predictive factors of RT-induced skin toxicities in early breast cancer patients treated with adjuvant RT after lumpectomy. Blood samples collected from patients enrolled in a prospective clinical study (n = 49) were analyzed at baseline and after the first delivered 2Gy RT dose. We designed an ex vivo culture system to differentiate patient blood monocytes into macrophages and treated them with M1 or M2-inducing cytokines before quantitative analysis of their "M1/M2" activation markers, iNOS, Arg1, and TGFß1. Statistical analysis was performed to correlate experimental data to clinical assessment of acute skin toxicity using Common Toxicity Criteria (CTC) grade for objective evaluation of skin reactions. Increased ARG1 mRNA significantly correlated with higher grades of erythema, moist desquamation, and CTC grade. Multivariate analysis revealed that increased ARG1 expression in macrophages after a single RT dose was an independent prognostic factor of erythema (p = 0 .032), moist desquamation (p = 0 .027), and CTC grade (p = 0 .056). Interestingly, multivariate analysis of ARG1 mRNA expression in macrophages stimulated with IL-4 also revealed independent prognostic value for predicting acute RT-induced toxicity factors, erythema (p = 0 .069), moist desquamation (p = 0 .037), and CTC grade (p = 0 .046). To conclude, our findings underline for the first time the biological significance of increased ARG1 mRNA levels as an early independent predictive biomarker of RT-induced acute skin toxicities.

Backbone assignment and secondary structure of the PLAT domain of human polycystin-1.

Polycystin-1 is a large transmembrane protein mutated in the common genetic disorder autosomal dominant polycystic kidney disease. One of the predicted intracellular domains of polycystin-1 is PLAT (Polycystin-1, Lipoxygenase and Alpha Toxin), which consists of 116 amino acids and is anchored to the membrane by linkers at both ends. It is predicted to have a large number of hydrophobic residues on the surface. Assignment of the NMR spectrum was hampered by considerable line broadening, and hence a programme of site-directed mutagenesis and searching for suitable solution conditions was undertaken. The optimum construct required fusion of the GB1 domain at the N-terminus and a His tag at the C-terminus, and proved to have several additional amino acids at both ends beyond the canonical domain boundaries, as well as mutation of W3128 to alanine. Optimum solubility required 500 mM sodium chloride, and usable spectra could only be obtained by perdeuteration. Backbone assignment was made using standard triple resonance spectra and is 88 % complete. The chemical shifts obtained suggest that a loop consisting of residues 3223-3228 is mobile in solution, and that the protein is similar in structure to a prediction produced by Swiss-Model based on the structure of a homologous protein.

Fabrication and luminescence of monolayered boron nitride quantum dots.

Monolayered boron nitride (BN) quantum dots (QDs; lateral size ≈10 nm) are fabricated using a novel method. Unlike monolayered BN sheets, these BN QDs exhibit blue-green luminescence due to defects formed during preparation. This optical behavior adds significant functionality to a material that is already receiving much attention. It is further shown that the QDs are nontoxic to biological cells and well suited to bio-imaging.

Cosegregation of focal segmental glomerulosclerosis in a family with familial partial lipodystrophy due to a mutation in LMNA.

BACKGROUND AND AIM: Focal segmental glomerulosclerosis (FSGS) is a common cause of idiopathic nephrotic syndrome in adults (35%). A number of genetic and familial forms of FSGS have been recognized. Here, we report a large pedigree with a pathogenic mutation in LMNA (R349W) in which four members were found to have biopsy-proven FSGS. The LMNA gene codes for lamins A and C, major components of the nuclear lamina which function in nuclear architecture, integrity and the regulation of gene expression. METHODS: Pedigree screening and mutation analysis of LMNA gene in all family members. Renal biopsies were performed in proteinuric patients. A molecular 3D model of the familial LMNA mutation was constructed. RESULTS: There were a total of 16 affected members from four generations, 12 of whom were found to carry the germline LMNA mutation. All affected adults had clinical features of familial partial lipodystrophy (FPLD) of the non-Dunnigan variety. Four patients within the same generation presented with a variable degree of renal impairment and proteinuria. Renal biopsies from all four revealed FSGS. The familial mutation is a missense change (R349W) in exon 6 of LMNA (c.1045C>T). CONCLUSIONS: We report a genetic link between LMNA and biopsy-proven FSGS in a large pedigree with FPLD. This unexpected association extends the disease spectrum of LMNA to the kidney and suggests that the physiological role of LMNA could be relevant to the maintenance of glomerular structure and function.

Spontaneous epithelial-mesenchymal transition and resistance to HER-2-targeted therapies in HER-2-positive luminal breast cancer.

Resistance to trastuzumab, a rationally designed HER-2-targeting antibody, remains a major hurdle in the management of HER-2-positive breast cancer. Preclinical studies suggest the mechanisms of trastuzumab resistance are numerous. Unfortunately, the majority of these studies are based around HER-2-positive (HER-2+) luminal cell lines. The role of epithelial to mesenchymal transition (EMT), a genetic program that confers a basal phenotype, may represent a novel mechanism of escape for HER-2+ luminal cells from trastuzumab treatment. Here we investigated this possibility using a model of clonal selection in HER-2+ luminal breast cancer cells. Following a random isolation and expansion of "colony clusters" from SKBR-3 cell lines, several colony clusters underwent a spontaneous EMT in-vitro. In addition to expression of conventional EMT markers, all mesenchymal colony clusters displayed a predominant CD44+/CD24- phenotype with decreased HER-2 expression and elevated levels of a ß1-integrin isoform with a high degree of N-glycosylation. Treatment with a ß1-integrin function-blocking antibody, AIIB2, preferentially decreased the N-glycosylated form of ß1-integrin, impaired mammosphere formation and restored epithelial phenotype in mesenchymal colony clusters. Using this model we provide the first clear evidence that resistance to trastuzumab (and lapatinib) can occur spontaneously as HER-2+ cells shift from a luminal to a basal/mesenchymal phenotype following EMT. While the major determinant of trastuzumab resistance in mesenchymal colony clusters is likely the down regulation of the HER-2 protein, our evidence suggests that multiple factors may contribute, including expression of N-glycosylated ß1-integrin.

Fabrication of luminescent monolayered tungsten dichalcogenides quantum dots with giant spin-valley coupling.

A high yield (>36 wt %) method has been developed of preparing monolayered tungsten dichalcogenide (WS2) quantum dots (QDs) with lateral size ∼8-15 nm from multilayered WS2 flakes. The monolayered WS2 QDs are, like monolayered WS2 sheets, direct semiconductors despite the flake precursors being an indirect semiconductor. However, the QDs have a significantly larger direct transition energy (3.16 eV) compared to the sheets (2.1 eV) and enhanced photoluminescence (PL; quantum yield ∼4%) in the blue-green spectral region at room temperature. UV/vis measurements reveal a giant spin-valley coupling of the monolayered WS2 QDs at around 570 meV, which is larger than that of monolayered WS2 sheets (∼400 meV). This spin-valley coupling was further confirmed by PL as direct transitions from the conduction band minimum to split valence band energy levels, leading to multiple luminescence peaks centered at around 369 (3.36 eV) and 461 nm (2.69 eV, also contributed by a new defect level). The discovery of giant spin-valley coupling and the strong luminescence of the monolayered WS2 QDs make them potentially of interests for the applications in semiconductor-based spintronics, conceptual valley-based electronics, quantum information technology and optoelectronic devices. However, we also demonstrate that the fabricated monolayered WS2 QDs can be a nontoxic fluorescent label for high contrast bioimaging application.

O6-Methylguanine-DNA methyltransferase is a novel negative effector of invasion in glioblastoma multiforme.

The dismal prognosis of glioblastoma multiforme (GBM) is mostly due to the high propensity of GBM tumor cells to invade. We reported an inverse relationship between GBM angiogenicity and expression of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), which has been extensively characterized for its role in resistance to alkylating agents used in GBM treatment. In the present study, given the major role of angiogenesis and invasion in GBM aggressiveness, we aimed to investigate the relationship between MGMT expression and GBM invasion. Stable overexpression of MGMT in the U87MG cell line significantly decreased invasion, altered expression of invasion-related genes, decreased expression of α(5)ß(1) integrin and focal adhesion kinase, and reduced their spindle-shaped morphology and migration compared with the empty vector control. Conversely, short hairpin RNA-mediated stable knockdown of MGMT or its pharmacologic depletion in the MGMT-positive T98G cell line were required for increased invasion. The inverse relationship between MGMT and invasion was further validated in primary GBM patient-derived cell lines. Using paraffin-embedded tumors from patients with newly diagnosed GBM (n = 59), tumor MGMT promoter hypermethylation (MGMT gene silencing) was significantly associated with increased immunohistochemical expression of the proinvasive matricellular protein secreted protein acidic and rich in cysteine (SPARC; P = 0.039, χ(2) test). Taken together, our findings highlight for the first time the role of MGMT as a negative effector of GBM invasion. Future studies are warranted to elucidate the role of SPARC in the molecular mechanisms underlying the inverse relationship between MGMT and GBM invasion and the potential use of MGMT and SPARC as biomarkers of GBM invasion.

A polycystin-2 (TRPP2) dimerization domain essential for the function of heteromeric polycystin complexes.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Earlier work has shown that PC1 and PC2 assemble into a polycystin complex implicated in kidney morphogenesis. PC2 also assembles into homomers of uncertain functional significance. However, little is known about the molecular mechanisms that direct polycystin complex assembly and specify its functions. We have identified a coiled coil in the C-terminus of PC2 that functions as a homodimerization domain essential for PC1 binding but not for its self-oligomerization. Dimerization-defective PC2 mutants were unable to reconstitute PC1/PC2 complexes either at the plasma membrane (PM) or at PM-endoplasmic reticulum (ER) junctions but could still function as ER Ca(2+)-release channels. Expression of dimerization-defective PC2 mutants in zebrafish resulted in a cystic phenotype but had lesser effects on organ laterality. We conclude that C-terminal dimerization of PC2 specifies the formation of polycystin complexes but not formation of ER-localized PC2 channels. Mutations that affect PC2 C-terminal homo- and heteromerization are the likely molecular basis of cyst formation in ADPKD.

MGMT modulates glioblastoma angiogenesis and response to the tyrosine kinase inhibitor sunitinib.

Angiogenesis inhibitors, such as sunitinib, represent a promising strategy to improve glioblastoma (GBM) tumor response. In this study, we used the O(6)-methylguanine methyltransferase (MGMT)-negative GBM cell line U87MG stably transfected with MGMT (U87/MGMT) to assess whether MGMT expression affects the response to sunitinib. We showed that the addition of sunitinib to standard therapy (temozolomide [TMZ] and radiation therapy [RT]) significantly improved the response of MGMT-positive but not of MGMT-negative cells. Gene expression profiling revealed alterations in the angiogenic profile, as well as differential expression of several receptor tyrosine kinases targeted by sunitinib. MGMT-positive cells displayed higher levels of vascular endothelial growth factor receptor 1 (VEGFR-1) compared with U87/EV cells, whereas they displayed decreased levels of VEGFR-2. Depleting MGMT using O(6)-benzylguanine suggested that the expression of these receptors was directly related to the MGMT status. Also, we showed that MGMT expression was associated with a dramatic increase in the soluble VEGFR-1/VEGFA ratio, thereby suggesting a decrease in bioactive VEGFA and a shift towards an antiangiogenic profile. The reduced angiogenic potential of MGMT-positive cells is supported by: (i) the decreased ability of their secreted factors to induce endothelial tube formation in vitro and (ii) their low tumorigenicity in vivo compared with the MGMT-negative cells. Our study is the first to show a direct link between MGMT expression and decreased angiogenicity and tumorigenicity of GBM cells and suggests the combination of sunitinib and standard therapy as an alternative strategy for GBM patients with MGMT-positive tumors.

Beta1-integrin circumvents the antiproliferative effects of trastuzumab in human epidermal growth factor receptor-2-positive breast cancer.

Resistance to trastuzumab, the monoclonal antibody targeting human epidermal growth factor receptor 2 (HER-2), is a major concern for HER-2-positive metastatic breast cancer (MBC) patients. To date, HER-2 status is the only available biomarker for selecting patients for trastuzumab-based therapy. Beta(1)-integrin, an adhesion molecule involved in cell survival and drug resistance, shares common downstream signaling elements with HER-2, such as the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase-1/2 (ERK1/2) pathways. The significance of beta(1)-integrin expression in HER-2-positive breast cancer and its involvement in a patient's response to trastuzumab-based therapy are unknown. We show here that overexpression of beta(1)-integrin is an independent negative prognostic factor for tumor progression of HER-2-positive MBC patients treated with trastuzumab-based chemotherapy. Enforced overexpression of beta(1)-integrin, its small interfering RNA-induced knockdown or treatment with a beta(1)-integrin-blocking antibody in HER-2-positive breast cancer cells, identified a strong inverse relationship between expression level of beta(1)-integrin and in vitro sensitivity to trastuzumab. Notably, beta(1)-integrin overexpression increased the phosphorylation of Akt-Ser473 and ERK1/2, thereby promoting survival and mitogenic signals to bypass the antiproliferative effects of trastuzumab. Our findings show that beta(1)-integrin provides a novel independent prognostic biomarker of trastuzumab response in HER-2-positive MBC patients and suggest a new target to augment the antiproliferative effects of trastuzumab.

Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs.

Caprin-1 is a ubiquitously expressed, well-conserved cytoplasmic phosphoprotein that is needed for normal progression through the G(1)-S phase of the cell cycle and occurs in postsynaptic granules in dendrites of neurons. We demonstrate that Caprin-1 colocalizes with RasGAP SH3 domain binding protein-1 (G3BP-1) in cytoplasmic RNA granules associated with microtubules and concentrated in the leading and trailing edge of migrating cells. Caprin-1 exhibits a highly conserved motif, F(M/I/L)Q(D/E)Sx(I/L)D that binds to the NTF-2-like domain of G3BP-1. The carboxy-terminal region of Caprin-1 selectively bound mRNA for c-Myc or cyclin D2, this binding being diminished by mutation of the three RGG motifs and abolished by deletion of the RGG-rich region. Overexpression of Caprin-1 induced phosphorylation of eukaryotic translation initiation factor 2alpha (eIF-2alpha) through a mechanism that depended on its ability to bind mRNA, resulting in global inhibition of protein synthesis. However, cells lacking Caprin-1 exhibited no changes in global rates of protein synthesis, suggesting that physiologically, the effects of Caprin-1 on translation were limited to restricted subsets of mRNAs. Overexpression of Caprin-1 induced the formation of cytoplasmic stress granules (SG). Its ability to bind RNA was required to induce SG formation but not necessarily its ability to enter SG. The ability of Caprin-1 or G3BP-1 to induce SG formation or enter them did not depend on their association with each other. The Caprin-1/G3BP-1 complex is likely to regulate the transport and translation of mRNAs of proteins involved with synaptic plasticity in neurons and cellular proliferation and migration in multiple cell types.