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Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine, whereas arginine is a context-dependent auxiliary sticker. In contrast, lysine weakens sticker-sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multichain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, and thus make the glycine versus serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation.

Liquid–liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation., Graphical Abstract Graphical AbstracthnRNPA1 phase separation is highly salt sensitive. Phase separation of the low-complexity domain (LCD) of hnRNPA1 increases with NaCl. In contrast, phase separation of full-length hnRNPA1 is salt-sensitive. At low NaCl concentrations, electrostatic RRM–LCD interactions occur and can contribute positively to phase separation, but they are screened at high NaCl concentrations. The folded domains solubilize hnRNPA1 under these conditions and prevent phase separation.

In vitro studies suggest that mapping the spatiotemporal complexity of nuclear factor κB (NF-κB) signaling is essential to understanding its function. The lack of tools to directly monitor NF-κB proteins in vivo has hindered such efforts. Here, we introduce reporter mice with the endogenous RelA (p65) or c-Rel labeled with distinct fluorescent proteins and a double knockin with both subunits labeled. Overcoming hurdles in simultaneous live-cell imaging of RelA and c-Rel, we show that quantitative features of signaling reflect the identity of activating ligands, differ between primary and immortalized cells, and shift toward c-Rel in microglia from aged brains. RelA:c-Rel heterodimer is unexpectedly depleted in the nuclei of stimulated cells. Trajectories of subunit co-expression in immune lineages reveal a reduction at key cell maturation stages. These results demonstrate the power of these reporters in gaining deeper insights into NF-κB biology, with the spectral complementarity of the labeled NF-κB proteins enabling diverse applications.

Not too sticky There is increasing evidence for a role of liquid-liquid phase separation (LLPS) in many cellular processes. Many proteins that undergo LLPS include prionlike domains (PLDs), which are enriched in polar amino acids and often interspersed with aromatic residues. Combining experimental data with simulations, Martin et al. quantified concentrations of PLDs in coexisting dilute and dense phases as a function of temperature and show that the phase behavior is determined by the number of aromatic residues and their patterning, with uniform patterning of aromatic residues promoting LLPS and inhibiting aggregation. They developed a sticker-and-spacers model that can predict the phase behavior of PLDs on the basis of their sequence. Science , this issue p. 694

Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular β-β interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular ‘Swiss Army Knives’ capable of providing protection through several different mechanisms simultaneously.

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging. Although challenging, characterizing the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with non-smFRET (NMR) data and reserved the smFRET data as an independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.

The condensation of biomolecules into biomolecular condensates via liquid-liquid phase separation (LLPS) is a ubiquitous mechanism that drives cellular organization. To enable these functions, biomolecules have evolved to drive LLPS and facilitate partitioning into biomolecular condensates. Determining the molecular features of proteins that encode LLPS will provide critical insights into a plethora of biological processes. Problematically, probing biomolecular dense phases directly is often technologically difficult or impossible. By capitalizing on the symmetry between the conformational behavior of biomolecules in dilute solution and dense phases, it is possible to infer details critical to phase separation by precise measurements of the dilute phase thus circumventing complicated characterization of dense phases. The symmetry between dilute and dense phases is found in the size and shape of the conformational ensemble of a biomolecule—parameters that small-angle X-ray scattering (SAXS) is ideally suited to probe. Recent technological advances have made it possible to accurately characterize samples of intrinsically disordered protein regions at low enough concentration to avoid interference from intermolecular attraction, oligomerization or aggregation, all of which were previously roadblocks to characterizing self-assembling proteins. Herein, we describe the pitfalls inherent to measuring such samples, the experimental details required for circumventing these issues and analysis methods that place the results of SAXS measurements into the theoretical framework of LLPS.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10 μl.

Intrinsically disordered protein regions (IDRs) — regions that do not fold into a fixed three-dimensional structure but instead exist in a heterogeneous ensemble of conformations — have recently entered mainstream cell biology in the context of liquid–liquid phase separation (LLPS). IDRs are frequently found to be enriched in phase-separated compartments. Due to this observation, the presence of an IDR in a protein is frequently assumed to be diagnostic of its ability to phase separate. In this review, we clarify the role of IDRs in biological assembly and explore the physical principles through which amino acids can confer the attractive molecular interactions that underlie phase separation. While some disordered regions will robustly drive phase separation, many others will not. We emphasize that rather than ‘disorder' driving phase separation, multivalency drives phase separation. As such, whether or not a disordered region is capable of driving phase separation will depend on the physical chemistry encoded within its amino acid sequence. Consequently, an in-depth understanding of that physical chemistry is a prerequisite to make informed inferences on how and why an IDR may be involved in phase separation or, more generally, in protein-mediated intermolecular interactions.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

The complex signaling dynamics of transcription factors can encode both qualitative and quantitative information about the extracellular environment, which increases the information transfer capacity and potentially supports accurate cellular decision-making. An important question is how these signaling dynamics patterns are translated into functionally appropriate gene regulation programs. To address this question for transcription factors of the nuclear factor κB (NF-κB) family, we profiled the single-cell dynamics of two major NF-κB subunits, RelA and c-Rel, induced by a panel of pathogen-derived stimuli in immune and nonimmune cellular contexts. Diverse NF-κB-activating ligands produced different patterns of RelA and c-Rel signaling dynamic features, such as variations in duration or time-integrated activity. Analysis of nascent transcripts delineated putative direct targets of NF-κB as compared to genes controlled by other transcriptional and posttranscriptional mechanisms and showed that the transcription of more than half of the induced genes was tightly linked to specific dynamic features of NF-κB signaling. Fibroblast and macrophage cell lines shared a cluster of such "NF-κB dynamics-decoding" genes, as well as cell type-specific decoding genes. Dissecting the subunit specificity of dynamics-decoding genes suggested that target genes were most often linked to both RelA and c-Rel or to RelA alone. Thus, our analysis reveals the cell type-specific interpretation of pathogenic information through the signaling dynamics of NF-κB.

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging. Although challenging, characterizing the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data used in their calculations. We used integrative modelling to understand and implement conformational restraints imposed by the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Forster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with non-smFRET (NMR) data and reserved the smFRET data as an independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the integrative ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling in calculating and drawing conclusions from IDP conformational ensembles.

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10 μl.

Liquid-liquid phase separation seems to play critical roles in the compartmentalization of cells through the formation of biomolecular condensates. Many proteins with low-complexity regions are found in these condensates, and they can undergo phase separation in vitro in response to changes in temperature, pH, and ion concentration. Low-complexity regions are thus likely important players in mediating compartmentalization in response to stress. However, how the phase behavior is encoded in their amino acid composition and patterning is only poorly understood. We discuss here that polymer physics provides a powerful framework for our understanding of the thermodynamics of mixing and demixing and for how the phase behavior is encoded in the primary sequence. We propose to classify low-complexity regions further into subcategories based on their sequence properties and phase behavior. Ongoing research promises to improve our ability to link the primary sequence of low-complexity regions to their phase behavior as well as the emerging miscibility and material properties of the resulting biomolecular condensates, providing mechanistic insight into this fundamental biological process across length scales.

Phosphatidic acid (PA), the central intermediate in membrane phospholipid synthesis, is generated by two acyltransferases in a pathway conserved in all life forms. The second step in this pathway is catalyzed by 1-acyl-sn-glycerol-3-phosphate acyltransferase, called PlsC in bacteria. Here we present the crystal structure of PlsC from Thermotoga maritima, revealing an unusual hydrophobic/aromatic N-terminal two-helix motif linked to an acyltransferase αβ-domain that contains the catalytic HX4D motif. PlsC dictates the acyl chain composition of the 2-position of phospholipids, and the acyl chain selectivity 'ruler' is an appropriately placed and closed hydrophobic tunnel. We confirmed this by site-directed mutagenesis and membrane composition analysis of Escherichia coli cells that expressed mutant PlsC. Molecular dynamics (MD) simulations showed that the two-helix motif represents a novel substructure that firmly anchors the protein to one leaflet of the membrane. This binding mode allows the PlsC active site to acylate lysophospholipids within the membrane bilayer by using soluble acyl donors.

Biomolecular condensates are emerging as an important organizational principle within living cells. These condensed states are formed by phase separation, yet little is known about how material properties are encoded within the constituent molecules and how the specificity for being in different phases is established. Here we use analytic theory to explain the phase behavior of the cancer-related protein SPOP and its substrate DAXX. Binary mixtures of these molecules have a phase diagram that contains dilute liquid, dense liquid, and gel states. We show that these discrete phases appear due to a competition between SPOP-DAXX and DAXX-DAXX interactions. The stronger SPOP-DAXX interactions dominate at sub-stoichiometric DAXX concentrations leading to the formation of crosslinked gels. The theory shows that the driving force for gel formation is not the binding energy, but rather the entropy of distributing DAXX molecules on the binding sites. At high DAXX concentrations the SPOP-DAXX interactions saturate, which leads to the dissolution of the gel and the appearance of a liquid phase driven by weaker DAXX-DAXX interactions. This competition between interactions allows multiple dense phases to form in a narrow region of parameter space. We propose that the molecular architecture of phase-separating proteins governs the internal structure of dense phases, their material properties and their functions. Analytical theory can reveal these properties on the long length and time scales relevant to biomolecular condensates.

Many cell signaling events are coordinated by intrinsically disordered protein regions (IDRs) that undergo multisite Serine/Threonine phosphorylation. The conformational properties of these IDRs prior to and following multi-site phosphorylation are directly relevant to understanding their functions. Here, we present results from biophysical studies and molecular simulations that quantify the conformational properties of an 81-residue IDR from the S. cerevisiae transcription factor Ash1. We show that the unphosphorylated Ash1 IDR adopts coil-like conformations that are expanded and well-solvated. This result contradicts inferences regarding global compaction that are derived from heuristics based on amino acid compositions for IDRs with low proline contents. Upon phosphorylation at ten distinct sites, the global conformational properties of pAsh1 are indistinguishable from those of unphosphorylated Ash1. This insensitivity derives from compensatory changes to the pattern of local and long-range intra-chain contacts. We show that the conformational properties of Ash1 and pAsh1 can be explained in terms of the linear sequence patterning of proline and charged residues vis-à-vis all other residues. The sequence features of the Ash1 IDR are shared by many other IDRs that undergo multisite phosphorylation. Accordingly, we propose that our findings might be generalizable to other IDRs involved in cell signaling.

Technological advances are continually improving our ability to obtain more accurate views about the inner workings of biological systems. One such rapidly evolving area is single cell biology, and in particular gene expression and its regulation by transcription factors in response to intrinsic and extrinsic factors. Regarding the study of transcription factors, we discuss some of the promises and pitfalls associated with investigating how individual cells regulate gene expression through modulation of transcription factor activities. Specifically, we discuss four leading experimental approaches, the data that can be obtained from each, and important considerations that investigators should be aware of when drawing conclusions from such data.

TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer9s disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS) in vitro , fluidizes liquid–liquid phase separated nuclear TDP‐43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.

Membrane receptor clustering and phase separation enable noise filtering in signaling

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent.

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. Background: PAR-2 is a protease-activated, G-protein-coupled receptor involved in inflammation, development, and immunity. Results: The GPI-anchored serine protease testisin activates PAR-2, inducing PAR-2 loss from the cell surface, internalization, and cellular signaling. Conclusion: PAR-2 activation by testisin reveals a unique mode of cell surface PAR-2 regulation. Significance: This is the first report of GPI-anchored protease-mediated cleavage and activation of a PAR.

Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis. KEY MESSAGES: Testisin inhibits metastatic ovarian tumor burden and ascites production. Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion. PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells.

Compromised gastrointestinal barrier function is strongly associated with the progressive and destructive pathologies of the two main forms of irritable bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD). Matriptase is a membrane-anchored serine protease encoded by suppression of tumorigenicity-14 (ST14) gene, which is critical for epithelial barrier development and homeostasis. Matriptase barrier-protective activity is linked with the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin, which is a co-factor for matriptase zymogen activation. Here we show that mRNA and protein expression of both matriptase and prostasin are rapidly down-regulated in the initiating inflammatory phases of dextran sulfate sodium (DSS)-induced experimental colitis in mice, and, significantly, the loss of these proteases precedes the appearance of clinical symptoms, suggesting their loss may contribute to disease susceptibility. We used heterozygous St14 hypomorphic mice expressing a promoter-linked β-gal reporter to show that inflammatory colitis suppresses the activity of the St14 gene promoter. Studies in colonic T84 cell monolayers revealed that barrier disruption by the colitis-associated Th2-type cytokines, IL-4 and IL-13, down-regulates matriptase as well as prostasin through phosphorylation of the transcriptional regulator STAT6 and that inhibition of STAT6 with suberoylanilide hydroxamic acid (SAHA) restores protease expression and reverses cytokine-induced barrier dysfunction. Both matriptase and prostasin are significantly down-regulated in colonic tissues from human subjects with active ulcerative colitis or Crohn's disease, implicating the loss of this barrier-protective protease pathway in the pathogenesis of irritable bowel disease.

In the Q(B) site of the Rhodobacter sphaeroides photosynthetic reaction center, the donation of a hydrogen bond from the hydroxyl group of Ser-L223 to the ubisemiquinone formed after the first flash is debatable. In this study, we use a combination of spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations to comprehensively explore this topic. We show that ENDOR, ESEEM, and HYSCORE spectroscopic differences between mutant L223SA and the wild-type sample (WT) are negligible, indicating only minor perturbations in the ubisemiquinone spin density for the mutant sample. Qualitatively, this suggests that a strong hydrogen bond does not exist in the WT between the Ser-L223 hydroxyl group and the semiquinone O(1) atom, as removal of this hydrogen bond in the mutant should cause a significant redistribution of spin density in the semiquinone. We show quantitatively, using QM/MM calculations, that a WT model in which the Ser-L223 hydroxyl group is rotated to prevent hydrogen bond formation with the O(1) atom of the semiquinone predicts negligible change for the L223SA mutant. This, together with the better agreement between key QM/MM calculated and experimental hyperfine couplings for the non-hydrogen-bonded model, leads us to conclude that no strong hydrogen bond is formed between the Ser-L223 hydroxyl group and the semiquinone O(1) atom after the first flash. The implications of this finding for quinone reduction in photosynthetic reaction centers are discussed.

In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (Q(A)) and secondary (Q(B)) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the Q(A) and Q(B) sites, using samples of (15)N-labeled reaction centers, with the native high spin Fe(2+) exchanged for diamagnetic Zn(2+), prepared in (1)H(2)O and (2)H(2)O solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the Q(A) SQ by Flores et al. (Biophys. J.2007, 92, 671-682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6-5.4 MHz. HYSCORE was then applied to the Q(B) SQ where we found proton lines corresponding to T ≈ 5.2, 3.7 MHz and T ≈ 1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/MM) calculations, employing a model of the Q(B) site, were used to assign the observed couplings to specific hydrogen bonding interactions with the Q(B) SQ. These calculations allow us to assign the T = 5.2 MHz proton to the His-L190 N(δ)H···O(4) (carbonyl) hydrogen bonding interaction. The T = 3.7 MHz spectral feature most likely results from hydrogen bonding interactions of O1 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the Q(B) site show less asymmetric distribution of unpaired spin density over the SQ than seen for the Q(A) site, consistent with available experimental data for (13)C and (17)O carbonyl hyperfine couplings. The implications of these interactions for Q(B) function and comparisons with the Q(A) site are discussed.

Liposomes are promising vehicles to deliver diagnostic and therapeutic agents to cells in vivo. After uptake into cells by endocytosis, liposomes are degraded in the endolysosomal system. Consequently, the encapsulated cargo molecules frequently remain sequestered in endosomal compartments; this limits their usefulness in many applications (e.g. gene delivery). To overcome this, various fusogenic peptides have been developed to facilitate delivery of liposomally-encapsulated molecules into the cytosol. One such peptide is the pH-sensitive influenza-derived peptide INF7. Liposomal delivery of imaging agents is an attractive approach for enabling cell imaging and cell tracking in vivo, but can be hampered by inadequate intracellular accumulation and retention of probes caused by exocytosis (and possible degradation) of endosome-entrapped probes. Such signal loss could be minimized by facilitating escape of probe molecules from endolysosomal compartments into the cytosol. We investigated the ability of co-encapsulated INF7 to release liposomally-delivered rhodamine fluorophores into the cytosol after endosomal acidification/maturation. We co-encapsulated INF7 and fluorescent rhodamine derivatives having vastly different transport properties to show that after endocytosis by CV1 cells, the INF7 peptide is activated by acidic endosomal pH and facilitates efficient release of the fluorescent tracers into the cytosol. Furthermore, we show that INF7-facilitated escape from endosomes markedly enhanced retention of tracers that cannot be actively extruded from the cytosol. Minimizing loss of intracellular probes improves cellular imaging by increasing the signal-to-noise ratio of images and lengthening the time window that imaging can be performed. In particular, this will enhance in vivo electron paramagnetic resonance imaging, an emergent magnetic resonance imaging modality requires exogenous paramagnetic imaging agents and is highly promising for cellular and molecular imaging.

Interactions involving intrinsically disordered proteins (IDPs) are crucial to a wide range of biological signaling and regulatory processes. Many of these interactions are characterized by a disorder-to-order transition upon binding. However, a new class of “fuzzy complexes” have emerged in which the IDP remains largely disordered. Such complexes are characterized by an IDP containing multiple weak binding motifs which individually interact with a binding partner in a dynamic equilibrium. The interaction of phophorylated Ash1, which participates in the regulation of mating type switching in yeast, with its ubiquitin ligase - SCFCdc4 -- is an example of a “fuzzy complex”. This work aims to define the interaction between the region of Ash1 spanning residues 420-500 which contains 10 suboptimal binding motifs for Cdc4, so-called Cdc4 phospho degrons (CPDs) with Cdc4. Ash1 is thought to behave similarly to the cyclin dependent kinase inhibitor Sic1, which requires a threshold level of phosphorylation to overcome electrostatic repulsion towards Cdc4. The polyelectrostatic interaction model implies that electrostatic averaging between rapidly exchanging bound states allows Cdc4 to sense the net charge of its disordered binding partner, which is modulated by phosphorylation.In order to probe the interaction and test the polyelectrostatic model, we use NMR spectroscopy, small angle x-ray scattering and fluorescence anisotropy binding experiments. All CPDs contained in Ash1 420-500 engage Cdc4, one at a time. SAXS indicates that the peptide is relatively compact. Fluorescence anisotropy measurements show the affinities of the entire Ash1 420-500 peptide and individual CPD motifs for Cdc4. These preliminary data are suggestive of a “fuzzy complex” and we are in the process of studying the phosphorylation dependence. The dynamic interaction suggests that Ash1 degradation must occur in an all-or-nothing fashion for biological function.

Avian Sarcoma and Leukosis Virus (ASLV) entry into cells proceeds through two major steps -priming by cognate receptors on the cell surface followed by low pH-induced fusion with endosomes. Subtype A viruses are able to utilize two naturally occurring isoforms of the TVA receptor for infection, the GPI-anchored (TVA800) and the transmembrane (TVA950) receptors, which are localized to different membrane domains. We monitored the consecutive steps of ASLV entry into cells expressing these receptor isoforms, including endocytosis, delivery into acidic compartments, and fusion with endosomes. Cells expressing TVA950 internalized ASLV much faster than those expressing the alternative receptor. In both cell lines, fusion occurred shortly after internalization, suggesting that virus uptake was the rate-limiting step of entry. We found that ASLV fusion with endosomes proceeded through two relatively long-lived intermediates, a hemifusion-like intermediate and a small fusion pore. The lifetime of these intermediates was dependant on TVA receptor isoforms and their expression levels. TVA950 supported more robust fusion intermediates compared to TVA800. These findings are consistent with ASLV trafficking by via TVA950 to endosomal compartments that are more conducive to fusion. Alternatively, transmembrane receptor might more efficiently prime the viral fusion proteins compared to the GPI-anchored receptor. This work has been supported by the NIH AI053668 grant.

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes., Author Summary Enveloped viruses are taken up into host cells after they bind to a specific receptor on the cell surface. These viruses are often delivered to specific compartments known as endosomes which have an acid environment that can trigger the virus and endosome membranes to fuse, allowing virus entry into the cell interior. In spite of extensive studies, key steps of this process are poorly understood. To examine the mechanism of virus-endosome fusion, we took advantage of a model retrovirus (ASLV). We imaged the entry of “labeled” ASLV into cells using fluorescence microscopy and found important differences between ASLV entry, depending upon whether the virus was attached to a transmembrane receptor or to a lipid-anchored receptor. A transmembrane receptor supported faster virus uptake followed by the formation of larger and more stable fusion pores, which was somewhat delayed relative to ASLV entry into acidic endosomes. This could be due, in part, to a relatively long-lived hemifusion intermediate that preceded the formation of a fusion pore in cells expressing a transmembrane, but not a lipid-anchored receptor. These findings shed light on functional consequences of virus entry via distinct endocytic routes and on critical fusion intermediates leading to infection.

Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-10 molecules functioning as primary (Q(A)) and secondary (Q(B)) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the Q(B) site semiquinone with nitrogens from the local protein environment in natural and (15)N uniformly labeled reactions centers. (14)N and (15)N HYSCORE spectra of the Q(B) semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from (14)N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. (15)N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (0.1 MHz). These results clearly indicate that the Q(B) semiquinone forms hydrogen bonds with two nitrogens and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the Q(B) site. On the basis of the quadrupole coupling constant, one nitrogen can only be assigned to N(delta) of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen.

3-pulse ESEEM and HYSCORE pulse sequences have been used to analyze the secondary electron acceptor semiquinone anion radical (QB-). Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone molecules functioning as primary and secondary electron acceptors. The primary quinone radical (QA-) has been extensively studied, and hydrogen bonds have been characterized at both carbonyls. The structure around QB- has received less attention. The O4 carbonyl has been suggested to be hydrogen bonded to the Nδ from a histidine at residue L190. The O1 carbonyl also possesses a hydrogen bond that is weaker than that at O4. OH from serine at L223 is important in the hydrogen bond structure at this carbonyl. However, contributions from surrounding peptide nitrogens are suggested by x-ray structures but have not yet been investigated by EPR methods. Pulsed EPR studies of the QB- radical confirm one strongly coupled nitrogen with NQI frequencies consistent with a histidine Nδ. 3-pulse ESEEM and HYSCORE spectra also contain peaks from a second nitrogen nucleus. A priori knowledge of the origins of these peaks is less clear, but could include contributions from a backbone nitrogen. Additionally, NQI modulation from 14N is sufficiently shallow to observe signals from 2 protons in HYSCORE measurements. These results suggest the possibility that an additional residue contributes to the stability of QB-. Supported by NSF grant MCB 08-18121.

The redox midpoint potential ( E m ) of Q A , the primary quinone of bacterial reaction centers, is substantially modulated by the protein environment. Quite subtle mutations in the Q A binding site, e.g., at residues M218, M252 and M265, cause significant increases in the equilibrium constant for electron transfer to Q B , which indicate relative lowering of the E m of Q A . However, reports of functional linkage between the Q A and Q B sites make it difficult to partition such effects between Q A and Q B from purely relative changes. We report here measurements on the yield of delayed fluorescence emission from the primary donor (P) accompanying the thermally activated charge recombination of P + Q A − to form the excited singlet state of the primary donor, P*. The results show that for mutations of the Q A site residues, Met M218 and Ile M265 , essentially all the substantial thermodynamic effect is localized at Q A , with no evidence for a significant effect of these residues on the properties of Q B or the mutual influence (linkage) of Q A and Q B . We also report a significant lowering of the E m of Q A by the native lipid, cardiolipin, which brings the E m in isolated reaction centers more in line with that seen in native membrane vesicles (chromatophores). Possible origins of this effect are discussed in the context of the Q A binding site structure.

The photosynthetic reaction center of Rhodobacter sphaeroides contains two identical ubiquinone molecules. The redox potentials of these two quinones are significantly different, and are tuned by the specific interactions with the protein environment of the binding pocket. The primary quinone (QA) has hydrogen bonds between the two carbonyls - O4 to the NδH of His M219 and O1 to the peptide NH of Ala M260. Small changes in this hydrogen bond structure could induce large changes to redox potential, as suggested by the effect of mutating the isoleucine at M265 to threonine, which induces a 100 mV change in the Em of QA. In order to better understand the nature of this redox tuning, M265 mutants have been analyzed with x-band ESEEM and HYSCORE. These techniques are very sensitive to the coupling of the semiquinone anion to surrounding nitrogen nuclei. The results show significant changes to the coupling between QA and nitrogens from both M260 and M219. Peaks associated with histidine Nδ show a substantially different NQI asymmetry parameter (η) than the wildtype. However, results from 15N labeled samples show that hyperfine coupling is fairly similar. This could suggest a rotation of the NQI tensor versus the g-tensor of the semiquinone, which could, in turn, indicate a large change of local electric field at the Nδ. Supported by NSF grant MCB 08-18121.