Cooperative Recruitment Research Articles

Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213’s dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.

Structural basis for transcription activation through cooperative recruitment of MntR.

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

Structural basis for transcription activation through cooperative recruitment of MntR.

The manganese transport regulator (MntR) from B. subtilis is a dual regulatory protein that responds to heightened Mn 2+ availability in the cell by both repressing the expression of uptake transporters and activating the expression of efflux proteins. Recent work indicates that, in its role as an activator, MntR binds several sites upstream of the genes encoding Mn 2+ exporters, leading to a cooperative response to manganese. Here, we use cryo-EM to explore the molecular basis of gene activation by MntR and report a structure of four MntR dimers bound to four 18-base pair sites across an 84-base pair regulatory region of the mneP promoter. Our structures, along with solution studies including mass photometry and in vivo transcription assays, reveal that MntR dimers employ polar and non-polar contacts to bind cooperatively to an array of low-affinity DNA-binding sites. These results reveal the molecular basis for cooperativity in the activation of manganese efflux.

Exploring university-industry collaboration in Vietnam: An in-depth review of types and influencing factors

This paper presents a comprehensive literature review on University-Industry Collaboration (UIC) in Vietnam, marking the first systematic examination of practice-based studies within the country. Our review aims to identify key forms and factors influencing UIC implementation in Vietnam, recommending future directions and highlighting key facilitators. Following the PRISMA 2009 guidelines, we identified 256 research papers across four databases (Web of Science, ERIC, ProQuest, and Google Scholar), ultimately selecting 25 papers after applying inclusion and exclusion criteria and conducting duplicate removal. Thematic and inductive qualitative content analysis methods were employed to address the review question, revealing observed forms of UIC in Vietnam, including sponsorship, internships, licensing, knowledge transfer, and cooperative recruitment. Influencing factors encompass 10 elements. Our review identifies deficiencies in UIC implementation and suggests potential areas for future development. These findings pave the way for exploring innovative UIC strategies amidst industrialization and globalization.

Systematic characterization of seed overlap microRNA cotargeting associated with lupus pathogenesis

BackgroundCombinatorial gene regulation by multiple microRNAs (miRNAs) is widespread and closely spaced target sites often act cooperatively to achieve stronger repression (“neighborhood” miRNA cotargeting). While miRNA cotarget sites are suggested to be more conserved and implicated in developmental control, the pathological significance of miRNA cotargeting remains elusive.ResultsHere, we report the pathogenic impacts of combinatorial miRNA regulation on inflammation in systemic lupus erythematosus (SLE). In the SLE mouse model, we identified the downregulation of two miRNAs, miR-128 and miR-148a, by TLR7 stimulation in plasmacytoid dendritic cells. Functional analyses using human cell lines demonstrated that miR-128 and miR-148a additively target KLF4 via extensively overlapping target sites (“seed overlap” miRNA cotargeting) and suppress the inflammatory responses. At the transcriptome level, “seed overlap” miRNA cotargeting increases susceptibility to downregulation by two miRNAs, consistent with additive but not cooperative recruitment of two miRNAs. Systematic characterization further revealed that extensive “seed overlap” is a prevalent feature among broadly conserved miRNAs. Highly conserved target sites of broadly conserved miRNAs are largely divided into two classes—those conserved among eutherian mammals and from human to Coelacanth, and the latter, including KLF4-cotargeting sites, has a stronger association with both “seed overlap” and “neighborhood” miRNA cotargeting. Furthermore, a deeply conserved miRNA target class has a higher probability of haplo-insufficient genes.ConclusionsOur study collectively suggests the complexity of distinct modes of miRNA cotargeting and the importance of their perturbations in human diseases.

Coupling of distant ATPase domains in the circadian clock protein KaiC.

The AAA+ family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.

ParB proteins can bypass DNA-bound roadblocks via dimer-dimer recruitment.

The ParABS system is essential for prokaryotic chromosome segregation. After loading at parS on the genome, ParB (partition protein B) proteins rapidly redistribute to distances of ~15 kilobases from the loading site. It has remained puzzling how this large-distance spreading can occur along DNA loaded with hundreds of proteins. Using in vitro single-molecule fluorescence imaging, we show that ParB from Bacillus subtilis can load onto DNA distantly of parS, as loaded ParB molecules themselves are found to be able to recruit additional ParB proteins from bulk. Notably, this recruitment can occur in cis but also in trans, where, at low tensions within the DNA, newly recruited ParB can bypass roadblocks as it gets loaded to spatially proximal but genomically distant DNA regions. The data are supported by molecular dynamics simulations, which show that cooperative ParB-ParB recruitment can enhance spreading. ParS-independent recruitment explains how ParB can cover substantial genomic distance during chromosome segregation, which is vital for the bacterial cell cycle.

The Evolution of Food Calls: Vocal Behaviour of Sooty Mangabeys in the Presence of Food.

The two main theories of food-associated calls in animals propose functions either in cooperative recruitment or competitive spacing. However, not all social animals produce food calls and it is largely unclear under what circumstances this call type evolves. Sooty mangabeys (Cercocebus atys) do not have food calls, but they frequently produce grunts during foraging, their most common vocalisation. We found that grunt rates were significantly higher when subjects were foraging in the group’s periphery and with small audiences, in line with the cooperative recruitment hypothesis. In a subsequent field experiment we presented highly desired food items and found that discovering individuals called, unless harassed by competitors, but that the calls never attracted others, confirming that the grunts do not convey any information referential to food. Our data thus suggest that the evolution of cooperative food calling is a two-step process, starting with increased motivation to vocalise in the feeding context, followed by the evolution of acoustic variants derived from context-general contact calls. This evolutionary transition may only occur in species that feed on clumped, high-quality resources where social feeding is competitive, a condition not met in sooty mangabeys.

Ribosomal DNA promoter recognition is determined in vivo by cooperation between UBTF1 and SL1 and is compromised in the UBTF-E210K neuroregression syndrome.

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

Cooperative recruitment of RDR6 by SGS3 and SDE5 during small interfering RNA amplification in Arabidopsis

Small interfering RNAs (siRNAs) are often amplified from transcripts cleaved by RNA-induced silencing complexes (RISCs) containing a small RNA (sRNA) and an Argonaute protein. Amplified siRNAs, termed secondary siRNAs, are important for reinforcement of target repression. In plants, target cleavage by RISCs containing 22-nucleotide (nt) sRNA and Argonaute 1 (AGO1) triggers siRNA amplification. In this pathway, the cleavage fragment is converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 6 (RDR6), and the dsRNA is processed into siRNAs by Dicer-like proteins. Because nonspecific RDR6 recruitment causes nontarget siRNA production, it is critical that RDR6 is specifically recruited to the target RNA that serves as a template for dsRNA formation. Previous studies showed that Suppressor of Gene Silencing 3 (SGS3) binds and stabilizes 22-nt sRNA-containing AGO1 RISCs associated with cleaved target, but how RDR6 is recruited to targets cleaved by 22-nt sRNA-containing AGO1 RISCs remains unknown. Here, using cell-free extracts prepared from suspension-cultured Arabidopsis thaliana cells, we established an in vitro system for secondary siRNA production in which 22-nt siRNA-containing AGO1-RISCs but not 21-nt siRNA-containing AGO1-RISCs induce secondary siRNA production. In this system, addition of recombinant Silencing Defective 5 (SDE5) protein remarkably enhances secondary siRNA production. We show that RDR6 is recruited to a cleavage fragment by 22-nt siRNA-containing AGO1-RISCs in coordination with SGS3 and SDE5. The SGS3-SDE5-RDR6 multicomponent recognition system and the poly(A) tail inhibition may contribute to securing specificity of siRNA amplification.

Epigenetic Mechanisms of HIV-1 Persistence.

Eradicating HIV-1 in infected individuals will not be possible without addressing the persistence of the virus in its multiple reservoirs. In this context, the molecular characterization of HIV-1 persistence is key for the development of rationalized therapeutic interventions. HIV-1 gene expression relies on the redundant and cooperative recruitment of cellular epigenetic machineries to cis-regulatory proviral regions. Furthermore, the complex repertoire of HIV-1 repression mechanisms varies depending on the nature of the viral reservoir, although, so far, few studies have addressed the specific regulatory mechanisms of HIV-1 persistence in other reservoirs than the well-studied latently infected CD4+ T cells. Here, we present an exhaustive and updated picture of the heterochromatinization of the HIV-1 promoter in its different reservoirs. We highlight the complexity, heterogeneity and dynamics of the epigenetic mechanisms of HIV-1 persistence, while discussing the importance of further understanding HIV-1 gene regulation for the rational design of novel HIV-1 cure strategies.

COOH-terminal SAA1 peptides fail to induce chemokines but synergize with CXCL8 and CCL3 to recruit leukocytes via FPR2

AbstractA natural leukocyte chemoattractant was isolated from bovine serum by an established 4-step purification procedure. Based on its relative molecular mass of 7287 and NH2-terminal sequence, the protein was identified as a carboxy-terminal peptide of the acute phase protein serum amyloid A1 (SAA1). This SAA1(46-112) fragment and its human equivalent SAA1(47-104) were chemically synthesized. Unlike intact SAA1α, these SAA fragments failed to directly chemoattract neutrophils and monocytes, to induce chemokines, and to stimulate downstream extracellular signal-regulated kinase signaling in monocytes. However, the SAA fragments potently synergized with CCL3 to induce monocyte migration and with CXCL8 to stimulate neutrophil shape changes and chemotaxis. Unlike intact SAA1α, SAA1(46-112) did not induce CXCL6 ex vivo but provoked a cooperative intraperitoneal neutrophil recruitment in mice when coinjected with CXCL6 into the peritoneal cavity. Moreover, SAA1(47-104) desensitized the synergy between intact SAA1α and CXCL8 in neutrophil chemotaxis, suggesting that this peptide binds formyl peptide receptor 2 (FPR2). This was evidenced by a complete blockade of synergy between the COOH-terminal SAA1 fragments and CXCL8 or CCL3 in neutrophil and monocyte chemotaxis, respectively, by the FPR2 antagonist WRW4. Thus, SAA1 is degraded into fragments lacking chemokine-inducing capacity, while keeping synergy with cytokine-induced chemokines to sustain limited inflammation.

The apoptosome molecular timer synergises with XIAP to suppress apoptosis execution and contributes to prognosticating survival in colorectal cancer

The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation.

Novel approach to therapeutic targeting of castration-resistant prostate cancer

Development of resistance to anti-androgen therapy limits the usefulness of second-generation androgen receptor (AR) antagonists including enzalutamide and abiraterone in castration resistant prostate cancer (CRPC) patients. Recent genomic studies reveal that AR-regulated genes contribute to CRPC emergence. Several reasons for the development of resistance towards anti-androgens have been hypothesized, including intracellular testosterone production, androgen overexpression, somatic mutations of AR resulting in a gain of function, constitutive activation of AR splice variants, imbalance in AR regulators, and bypass of AR in CRPC progression. Recent findings suggest that epigenetic alterations are involved in the deregulation of AR signaling. Overexpression of enhancer of zeste homolog 2 (EZH2), the enzymatic member of the polycomb repressor complex PRC2, has emerged as a key activator of AR in CRPC. Studies indicate that overabundance of EZH2 in localized prostate tumors increases the risk of biochemical recurrence after surgery, as it activates AR by enhancing methylation, resulting in the suppression of tumor suppressor genes and activation of oncogenes. This apparent association between EZH2 and AR in activating target genes by cooperative recruitment might play a critical role in the emergence of CRPC. Our hypothesis is that combination treatment targeting EZH2 and AR may be a novel efficacious therapeutic regime for the treatment of castrate resistant prostate cancer, and we propose to investigate this possibility.

Cooperative recruitment of Arl4A and Pak1 to the plasma membrane contributes to sustained Pak1 activation for cell migration.

Cell migration requires the coordination of multiple signaling pathways involved in membrane dynamics and cytoskeletal rearrangement. The Arf-like small GTPase Arl4A has been shown to modulate actin cytoskeleton remodeling. However, evidence of the function of Arl4A in cell migration is insufficient. Here, we report that Arl4A acts with the serine/threonine protein kinase Pak1 to modulate cell migration through their cooperative recruitment to the plasma membrane. We first observed that Arl4A and its isoform Arl4D interact with Pak1 and Pak2 and showed that Arl4A recruits Pak1 and Pak2 to the plasma membrane. The fibronectin-induced Pak1 localization at the plasma membrane is reduced in Arl4A-depleted cells. Unexpectedly, we found that Pak1, but not Arl4A-binding-defective Pak1, can recruit a cytoplasmic myristoylation-deficient Arl4A-G2A mutant to the plasma membrane. Furthermore, we found that the Arl4A-Pak1 interaction, which is independent of Rac1 binding to Pak1, is required for Arl4A-induced cell migration. Thus, we infer that there is feedback regulation between Arl4A and Pak1, in which they mutually recruit each other to the plasma membrane for Pak1 activation, thereby modulating cell migration through direct interaction.

Dendritic NMDA receptors in parvalbumin neurons enable strong and stable neuronal assemblies.

Parvalbumin-expressing (PV+) GABAergic interneurons mediate feedforward and feedback inhibition and have a key role in gamma oscillations and information processing. The importance of fast synaptic recruitment and action potential initiation and repolarization, and rapid synchronous GABA release by PV+ cells, is well established. In contrast, the functional significance of PV+ cell NMDA receptors (NMDARs), which generate relatively slow postsynaptic currents, is unclear. Underlining their potential importance, several studies implicate PV+ cell NMDAR disruption in impaired network function and circuit pathologies. Here, we show that dendritic NMDARs underlie supralinear integration of feedback excitation from local pyramidal neurons onto mouse CA1 PV+ cells. Furthermore, by incorporating NMDARs at feedback connections onto PV+ cells in spiking networks, we show that these receptors enable cooperative recruitment of PV+ interneurons, strengthening and stabilising principal cell assemblies. Failure of this phenomenon provides a parsimonious explanation for cognitive and sensory gating deficits in pathologies with impaired PV+ NMDAR signalling.

Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature

Heart failure is a life-threatening condition that occurs when the heart muscle becomes weakened and cannot adequately circulate blood and nutrients around the body. Omecamtiv mecarbil (OM) is a compound that has been developed to treat systolic heart failure via targeting the cardiac myosin heavy chain to increase myocardial contractility. Biophysical and biochemical studies have found that OM increases calcium (Ca2+) sensitivity of contraction by prolonging the myosin working stroke and increasing the actin-myosin cross-bridge duty ratio. Most in vitro studies probing the effects of OM on cross-bridge kinetics and muscle force production have been conducted at subphysiological temperature, even though temperature plays a critical role in enzyme activity and cross-bridge function. Herein, we used skinned, ventricular papillary muscle strips from rats to investigate the effects of [OM] on Ca2+-activated force production, cross-bridge kinetics, and myocardial viscoelasticity at physiological temperature (37°C). We find that OM only increases myocardial contractility at submaximal Ca2+ activation levels and not maximal Ca2+ activation levels. As [OM] increased, the kinetic rate constants for cross-bridge recruitment and detachment slowed for both submaximal and maximal Ca2+-activated conditions. These findings support a mechanism by which OM increases cardiac contractility at physiological temperature via increasing cross-bridge contributions to thin-filament activation as cross-bridge kinetics slow and the duration of cross-bridge attachment increases. Thus, force only increases at submaximal Ca2+ activation due to cooperative recruitment of neighboring cross-bridges, because thin-filament activation is not already saturated. In contrast, OM does not increase myocardial force production for maximal Ca2+-activated conditions at physiological temperature because cooperative activation of thin filaments may already be saturated.

Natural Genetic Variation Reveals Key Features of Epigenetic and Transcriptional Memory in Virus-Specific CD8 T Cells

Stable changes in chromatin states and gene expression in cells of the immune system form the basis for memory of infections and other challenges. Here, we used naturally occurring cis-regulatory variation in wild-derived inbred mouse strains to explore the mechanisms underlying long-lasting versus transient gene regulation in CD8 Tcells responding to acute viral infection. Stably responsive DNA elements were characterized by dramatic and congruent chromatin remodeling events affecting multiple neighboring sites and required distinct transcription factor (TF) binding motifs for their accessibility. Specifically, we found that cooperative recruitment of T-box and Runx family transcription factors to shared targets mediated stable chromatin remodeling upon Tcell activation. Our observations provide insights into the molecular mechanisms driving virus-specific CD8 Tcell responses and suggest a general mechanism for the formation of transcriptional and epigenetic memory applicable to other immune and non-immune cells.

Abstract 4815: Efficacy and toxicity of combinatorial therapy with EZH2 and androgen receptor inhibitor for castration-resistant prostate cancer

Abstract Enhancer of zeste homolog 2 (EZH2), the enzymatic member of the polycomb repressor complex PRC2 has emerged as a key activator of androgen receptor in castration-resistant prostate cancer (CRPC). Androgen deprivation therapy (ADT) is a main therapeutic modality for advance-stage metastatic prostate cancer targeting AR. Recent studies shed light on the fact that overabundance of EZH2 in localized cancers increases aggressiveness and their recurrence. EZH2 activates AR through methylation-mediated gene suppression. Though prostate cancers respond to ADT treatment initially they subsequently re-emerge as CRPC. In recent years, Enzalutamide (ENZU), a second generation AR antagonist exhibits survival advantage in CRPC patients, but show relapse within a year, activating AR in these tumors. Recent genomic studies reveal AR-regulated genes contribute to CRPC emergence. This apparent association between EZH2 and AR in activating target genes by cooperative recruitment may lead to the development of chemoresistance. Therefore we rationalize that combination treatment targeting EZH2 and AR could be efficacious and significantly inhibit proliferation and metastasis of CRPC cells. Human prostate cancer C4-2B and 22Rv1, representative of CRPC tumors, were treated with EZH2 inhibitor GSK126 and AR antagonist (ENZU) individually and in combination, followed by assessment of cell viability, cell cycle analysis, migration, invasion and expression of various target genes by Western blotting. ELISA was employed to assess the change in activity of histone methylation in these cells. Treatment of C4-2B and 22Rv1 cells individually with GSK126 and ENZU (2.5-80 µM) for 24 h exhibited a partial suppressive effect in cell growth. Using GSK126 and ENZU combination at 1:1, 1:5, 1:10 and 1:20 micro-molar ratio exhibited increased cell growth inhibition, where 1:10 ratio showed synergistic effect in inhibiting cell growth in both cell lines. This combination caused marked increase in G0/G1-phase cell cycle arrest followed by cell death, inhibition of migration and invasion in both cell lines. Furthermore, combination treatment led to significant reduction in the protein expression of AR, AR-V7, EZH2, SUZ12, EED, p-Akt (Ser473) in both cell lines, compared to individual treatments. Combined treatment also caused significant decrease in the levels of H3K27 methylation and its activity. Taken together, our results demonstrate that simultaneous inhibition of AR and EZH2 is more efficacious in inhibiting the growth of CRPC cells. This opens new possibilities of uncovering a novel path of treating CRPC by simultaneously targeting EZH2 and AR using a combinatorial approach. Citation Format: Eswar Shankar, Daniel Franco, Omair Iqbal, Stephen Moreton, Rajnee Kanwal, Sanjay Gupta. Efficacy and toxicity of combinatorial therapy with EZH2 and androgen receptor inhibitor for castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4815.

A novel fluorescence microscopic approach to quantitatively analyse protein-induced membrane remodelling

Membrane remodelling or the bending and rupture of the lipid bilayer occurs during diverse cellular processes such as cell division, synaptic transmission, vesicular transport, organelle biogenesis and sporulation. These activities are brought about by the localized change in membrane curvature, which in turn causes lipid-packing stress, of a planar lipid bilayer by proteins. For instance, vesicular transport processes are typically characterized by the cooperative recruitment of proteins that induce budding of a planar membrane and catalyse fission of the necks of membrane buds to release vesicles. The analysis of such membrane remodelling reactions has traditionally been restricted to electron microscopy-based approaches or force spectroscopic analysis of membrane tethers pulled from liposome-based model membrane systems. Our recent work has demonstrated the facile creation of tubular model membrane systems of supported membrane tubes (SMrTs), which mimic late-stage intermediates of typical vesicular transport reactions. This review addresses the nature of such an assay system and a fluorescence-intensity-based analysis of changes in tube dimensions that is indicative of the membrane remodelling capacity of proteins.