Cell Penetration Profiling Using the Chloroalkane Penetration Assay.

Biotherapeutics are a promising class of molecules in drug discovery, but they are often limited to extracellular targets due to their poor cell penetration. High-throughput cell penetration assays are required for the optimization of biotherapeutics for enhanced cell penetration. We developed a HaloTag-based assay called the chloroalkane penetration assay (CAPA), which is quantitative, high-throughput, and compartment-specific. We demonstrate the ability of CAPA to profile extent of cytosolic penetration with respect to concentration, presence of serum, temperature, and time. We also used CAPA to investigate structure-penetration relationships for bioactive stapled peptides and peptides fused to cell-penetrating sequences. CAPA is not only limited to measuring cytosolic penetration. Using a cell line where HaloTag is localized to the nucleus, we show quantitative measurement of nuclear penetration. Going forward, CAPA will be a valuable method for measuring and optimizing the cell penetration of biotherapeutics.

Diversity-Oriented Stapling Yields Intrinsically Cell-Penetrant Inducers of Autophagy.

Autophagy is an essential pathway by which cellular and foreign material are degraded and recycled in eukaryotic cells. Induction of autophagy is a promising approach for treating diverse human diseases, including neurodegenerative disorders and infectious diseases. Here, we report the use of a diversity-oriented stapling approach to produce autophagy-inducing peptides that are intrinsically cell-penetrant. These peptides induce autophagy at micromolar concentrations in vitro, have aggregate-clearing activity in a cellular model of Huntington's disease, and induce autophagy in vivo. Unexpectedly, the solution structure of the most potent stapled peptide, DD5-o, revealed an α-helical conformation in methanol, stabilized by an unusual (i,i+3) staple which cross-links two d-amino acids. We also developed a novel assay for cell penetration that reports exclusively on cytosolic access and used it to quantitatively compare the cell penetration of DD5-o and other autophagy-inducing peptides. These new, cell-penetrant autophagy inducers and their molecular details are critical advances in the effort to understand and control autophagy. More broadly, diversity-oriented stapling may provide a promising alternative to polycationic sequences as a means for rendering peptides more cell-penetrant.

C-terminal variable AGES domain of Thymosin ß4: the molecule's primary contribution in support of post-ischemic cardiac function and repair.

Repairing defective cardiac cells is important towards improving heart function. Due to the frequency and severity of ischemic heart disease, management of patients featuring this type of cardiac failure receives significant interest. Previously we discovered that Thymosin ß4 (TB4), a 43 amino-acid secreted actin sequestering peptide, is beneficial for myocardial cell survival and coronary re-growth after infarction in adult mammals. Considering the regenerative potential of full-length TB4 in the heart, and that minimal structural variations alter TB4's influence on actin assembly and cell movement, we investigated how various TB4 domains affect cardiac cell behavior and post-ischemic mammalian heart function. We synthesized 17 domain combinations of full-length TB4 and analyzed their impact on embryonic cardiac cells in vitro, and after cardiac infarction in vivo. We discovered the domains of TB4 affect cardiac cell behavior distinctly. We revealed TB4 specific C-terminal tetrapeptide, AGES, increases embryonic cardiac cell migration and myocyte beating in culture, and improves adult mammalian heart function following ischemia. Investigating the molecular background and mechanism we discovered systemic injection of AGES enhances early myocyte survival by activating Akt-mediated signaling mechanisms, increases coronary vessel growth and inhibits inflammation in mice and pigs. Biodistribution analyses revealed cardiomyocytes uptake AGES efficiently in vitro and in vivo projecting a potential independent clinical utilization for the tetrapeptide. Our comprehensive domain investigations also suggest, preservation and/or restoration of cardiomyocyte communication is a target of TB4 and AGES, and critical to improve post-ischemic heart function in pigs. In summary, we identified the C-terminal four amino-acid variable end of TB4 as the essential and responsible domain for the molecule's full benefits in the hypoxic heart. Additionally, we introduced AGES as a novel, systemically applicable drug candidate to aid cardiac infarction in adult mammals.

PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution.

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.

Phosphorylation of the spindle checkpoint protein Mad2 regulates its conformational transition.

Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2(S195D) mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2(S195D) fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2(S195D) in human cells causes checkpoint defects. Our results indicate that Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.

Orexin A, an amphipathic α-helical neuropeptide involved in pleiotropic functions in the nervous and immune systems: Synthetic approach and biophysical studies of the membrane-bound state.

This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease.

The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase-1 and blocks mitotic progression in bloodstream forms.

Aurora kinase family members co-ordinate a range of events associated with mitosis and cytokinesis. Anti-cancer therapies are currently being developed against them. Here, we evaluate whether Aurora kinase-1 (TbAUK1) from pathogenic Trypanosoma brucei might be targeted in anti-parasitic therapies as well. Conditional knockdown of TbAUK1 within infected mice demonstrated its essential contribution to infection. An in vitro kinase assay was developed which used recombinant trypanosome histone H3 as a substrate. Tandem mass spectroscopy identified a novel phosphorylation site in the carboxyl-tail of recombinant trypanosome histone H3. Hesperadin, an inhibitor of human Aurora B, prevented the phosphorylation of substrate with IC(50) of 40 nM. Growth of cultured bloodstream forms was also sensitive to Hesperadin (IC(50) of 50 nM). Hesperadin blocked nuclear division and cytokinesis but not other aspects of the cell cycle. Consequently, growth arrested cells accumulated multiple kinetoplasts, flagella and nucleoli, similar to the effects of RNAi-dependent knockdown of TbAUK1 in cultured bloodstream forms cells. Molecular models predicted high-affinity binding of Hesperadin to both conserved and novel sites in TbAUK1. Collectively, these data demonstrate that cell cycle progression is essential for infections with T. brucei and that parasite Aurora kinases can be targeted with small-molecule inhibitors.

Author Correction: Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.

Manipulating cell migration and proliferation with a light-activated polypeptide.

Remote control of cells: A polypeptide has been made that stimulates proliferation and migration of cells upon photochemical activation. This light-activated polypeptide enables spatially defined control of cell populations at the scale of tissue organization; this is accomplished without physically contacting the cells or modifying their substrate. Polypeptide growth and differentiation factors modulate a wide variety of cell behaviors and can be used to manipulate cells in vitro for tissue engineering and basic studies of cell biology. To emulate in vitro the spatial aspect of growth factor function, new methods are needed to generate defined spatial gradients of activity. Polypeptide factors that are engineered to be activated with light provide a method for creating concentration gradients with the fine precision in space and time with which light can be directed. As a first test of this approach, we have chemically synthesized a polypeptide with the sequence of epidermal growth factor in which a critical glutamate is "caged" with a photoremovable group. Photolysis of this polypeptide afforded maximal mitogenic and chemokinetic activity at concentrations at which the caged factor was inactive. Spatially resolved photolysis of the factor resulted in spatial patterning of fibroblasts. This system will be useful for ex vivo tissue engineering and for investigating the interactions of cells with their matrix and the role of chemical gradients in biological pattern formation.

Novel reversible biotinylated probe for the selective enrichment of phosphorylated peptides from complex mixtures.

To improve the detection of phosphorylated peptides/proteins, we developed a novel protocol that involves the chemical derivatization of phosphate groups with a chemically engineered biotinylated-tag (biotin-tag), possessing three functional domains; a biotin group for binding to avidin, a base-labile 4-carboxy fluorenyl methoxycarbonyl (4-carboxy Fmoc) group, and a nucleophilic sulfhydryl moiety on the side-chain of cysteine. Using this approach, the derivatized, enzymatically digested peptides were selectively separated from unrelated sequences and impurities on immobilized avidin. Unlike previously published phosphopeptide enrichment procedures, this approach upon treatment with mild base liberates a covalently bound Gly-Cys analog of the peptide(s) of interest, exhibiting improved RP-HPLC retention and MS ionization properties compared with the precursor phosphopeptide sequence. The results obtained for a model peptide Akt-1 and two protein digests, demonstrated that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of fmol/microL.

Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag.

To improve the detection of phosphorylated peptides/proteins, a combination of optimized MS-based strategies were used involving chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a nanoscale Ni(2+)-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot beta-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni(2+)-IMAC and released using either imidazole or cleavage with Factor Xa. This novel capture and enzyme-mediated release provided an additional element of selectivity and yielded phosphopeptide-specific modifications with enhanced MS ionization characteristics. The eluted peptides were mapped using MALDI-TOF MS and QTRAP ESI-MS/MS techniques. The results obtained for a model peptide and two tryptic protein digests show that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles per microliter.

Mass spectrometry analysis of 2-nitrophenylhydrazine carboxy derivatized peptides.

Peptides with two or more basic residues, including those with post-translational modifications (PTMs), such as methylation and phosphorylation, can be highly hydrophilic and, therefore, are often difficult to be retained on a reversed-phase (RP) column. In addition, these highly hydrophilic peptides may carry two or more positive charges, which often fragment poorly upon collisionally activated dissociation (CAD), resulting in few sequence-specific ions. C-terminal rearrangement may also occur during CAD. Furthermore, some PTMs are labile and tend to be lost when subjected to CAD as is the case with phosphorylation on serine or threonine. To overcome the difficulties of separation, detection, and fragmentation of highly hydrophilic peptides, we report here the effect of carboxy group derivatization with 2-nitrophenylhydrazine (this strategy will be called NPHylation for simplicity). NPHylation significantly increases the hydrophobicity of the peptides, eliminates C-terminal rearrangement in all cases, and offers enhanced sensitivity in some cases. In addition, the CAD spectra of the resulting NPHylated peptides carry more sequence-specific ions due to significant reduction of sequence scrambling as observed for peptide EHAGVISVL. Furthermore, the different carboxy derivatives of this peptide undergo sequence scrambling to varying degrees, which clearly demonstrates that the C-terminus has a profound effect on peptide fragmentation. Finally, sequence scrambling is a charge dependent phenomenon, which affects CAD of doubly charged peptides far more than their singly charged counterparts.

Characterization of AMPylation on threonine, serine, and tyrosine using mass spectrometry.

Recent studies have suggested that adenosine 5'-monophosphate (AMP) post-translational modification of proteins could represent a novel molecular signaling pathway. Mass spectrometric fragmentation characteristics of this modification have not previously been described and studied systematically. In this work, we therefore examined the fragmentation pattern of chemically synthesized peptides containing AMPylated Thr, Ser, and Tyr. The formation of characteristic ions and the influence of collision energy (CE) on the detection of characteristic ions and their relative peak intensity are reported. When peptide with AMPylated Ser/Thr underwent collision induced dissociation (CID), peaks at m/z 348.1, 136.1, and 250.1, fragments with AMP group attached, and fragments consistent with neutral loss of 347 Da were major characteristic ions; fragments consistent with neutral loss of 135 Da or 249 Da were weaker and not always detectable. The observations for Tyr AMPylation followed the same general patterns as those for Ser/Thr modification, with the exception that the ions detected for Tyr AMPylation did not include either the peak at m/z 348.1, or fragments with a mass shift of -347 Da. The results described in this paper highlight a series of diagnostic ions, which can be used not only to confidently identify the AMPylation site based on MS and MS/MS data, but also to selectively scan AMPylated peptides in complex protein mixtures.

Characterization of a rabbit polyclonal antibody against threonine-AMPylation.

An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins.

AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling.

The Vibrio parahaemolyticus type III effector VopS is implicated in cell rounding and the collapse of the actin cytoskeleton by inhibiting Rho guanosine triphosphatases (GTPases). We found that VopS could act to covalently modify a conserved threonine residue on Rho, Rac, and Cdc42 with adenosine 5'-monophosphate (AMP). The resulting AMPylation prevented the interaction of Rho GTPases with downstream effectors, thereby inhibiting actin assembly in the infected cell. Eukaryotic proteins were also directly modified with AMP, potentially expanding the repertoire of posttranslational modifications for molecular signaling.

Structural basis for the inhibition of the LSD1 histone demethylase by the antidepressant trans-2-phenylcyclopropylamine.

Histone modifications, such as acetylation and methylation, are important epigenetic marks that regulate diverse biological processes that use chromatin as the template, including transcription. Dysregulation of histone acetylation and methylation leads to the silencing of tumor suppressor genes and contributes to cancer progression. Inhibitors of enzymes that catalyze the addition and removal of these epigenetic marks thus have therapeutic potential for treating cancer. Lysine-specific demethylase 1 (LSD1) is the first discovered histone lysine demethylase and, with the help of its cofactor CoREST, specifically demethylates mono- and dimethylated histone H3 lysine 4 (H3-K4), thus repressing transcription. Because LSD1 belongs to the family of flavin adenine dinucleotide (FAD)-dependent amine oxidases, certain inhibitors of monoamine oxidases (MAOs), including the clinically used antidepressant trans-2-phenylcyclopropylamine (PCPA; tranylcypromine; Parnate), are also capable of inhibiting LSD1. In this study, we have further measured the kinetic parameters of the inhibition of LSD1 by PCPA and determined the crystal structure of LSD1-CoREST in the presence of PCPA. Our structural and mass spectrometry analyses are consistent with PCPA forming a covalent adduct with FAD in LSD1 that is distinct from the FAD-PCPA adduct of MAO B. The structure also reveals that the phenyl ring of the FAD-PCPA adduct in LSD1 does not form extensive interactions with active-site residues. This study thus provides the basis for designing more potent inhibitors of LSD1 that contain substitutions on the phenyl ring of PCPA to fully engage neighboring residues.

Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation.

Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor kappaB (NFkappaB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl-coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.

Solid-state NMR structural studies of the fibril form of a mutant mouse prion peptide PrP89-143(P101L).

The peptide fragment 89-143 of the prion protein (carrying a P101L mutation) is biologically active in transgenic mice when in a fibrillar form. Injection of these fibrils into transgenic mice (expressing full length PrP with the P101L mutation) induces a neurodegenerative prion disease (Kaneko et al., J. Mol. Biol. 295 (2000) 997). Here we present solid-state NMR studies of PrP(89-143)(P101L) fibrils, probing the conformation of residues in the hydrophobic segment 112-124 with chemical shifts. The conformations of glycine residues were analyzed using doubly (13)C=O labeled peptides by two-dimensional (2D) double-quantum correlation, and double-quantum filtered dephasing distance measurements. MQ-NMR experiments were carried out to probe the relative alignment of the individual peptides fibrils. These NMR studies indicate that the 112-124 segment adopts an extended beta-sheet conformation, though not in a parallel, in register alignment. There is evidence for conformational variability at Gly 113. DQ correlation experiments provide useful information in regions with conformational heterogeneity.

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains.

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP(Sc)(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4 degrees C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP(Sc)(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a beta-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP(Sc)(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP(Sc) in the brains of Tg196 mice. Our results argue that MoPrP(Sc)(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP(C)(P101L) as well as in Tg196 mice expressing low levels of MoPrP(C)(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a beta-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.