Small Effector Molecules Research Articles

Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of β1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration. Other cellular defects in presence of GNE mutation include altered ER redox state and chaperone expression such as HSP70 or PrdxIV. Currently, there is no cure to treat GNEM. Possible therapeutic trials focus on supplementation with sialic acid, ManNAc, sialyllactose and gene therapy that slows the disease progression. In the present study, we analyzed the effect of small molecules like BGP-15 (HSP70 modulator), IGF-1 (IGF-1R ligand) and CGA (cofilin activator) on cellular phenotypes of GNE heterozygous knock out L6 rat skeletal muscle cell line (SKM‑GNEHz). Treatment with BGP-15 improved GNE epimerase activity by 40 % and reduced ER stress by 45 % for SKM‑GNEHz. Treatment with IGF-1 improved epimerase activity by 37.5 %, F-actin assembly by 100 %, cell migration upto 36 % (36 h) and atrophy by 0.44-fold for SKM‑GNEHz. Treatment with CGA recovered epimerase activity by 49 %, F-actin assembly by 132 % and cell migration upto 41 % (24 h) in SKM‑GNEHz. Our study shows that treatment with these small effector molecules reduces the detrimental phenotype observed in SKM‑GNEHz, thereby, providing insights into potential therapeutic targets for GNEM.

Cultured Vagal Afferent Neurons as Sensors for Intestinal Effector Molecules.

The gut-brain axis embodies the bi-directional communication between the gastrointestinal tract and the central nervous system (CNS), where vagal afferent neurons (VANs) serve as sensors for a variety of gut-derived signals. The gut is colonized by a large and diverse population of microorganisms that communicate via small (effector) molecules, which also act on the VAN terminals situated in the gut viscera and consequently influence many CNS processes. However, the convoluted in vivo environment makes it difficult to study the causative impact of the effector molecules on VAN activation or desensitization. Here, we report on a VAN culture and its proof-of-principle demonstration as a cell-based sensor to monitor the influence of gastrointestinal effector molecules on neuronal behavior. We initially compared the effect of surface coatings (poly-L-lysine vs. Matrigel) and culture media composition (serum vs. growth factor supplement) on neurite growth as a surrogate of VAN regeneration following tissue harvesting, where the Matrigel coating, but not the media composition, played a significant role in the increased neurite growth. We then used both live-cell calcium imaging and extracellular electrophysiological recordings to show that the VANs responded to classical effector molecules of endogenous and exogenous origin (cholecystokinin serotonin and capsaicin) in a complex fashion. We expect this study to enable platforms for screening various effector molecules and their influence on VAN activity, assessed by their information-rich electrophysiological fingerprints.

Functional characterization of GNE mutations prevalent in Asian subjects with GNE myopathy, an ultra-rare neuromuscular disorder

UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) is a bifunctional enzyme (N-terminal epimerase and C-terminal Kinase domain) that catalyses the rate limiting step in sialic acid biosynthesis. More than 200 homozygous missense or compound heterozygous mutations in GNE have been reported worldwide to cause a rare neuromuscular disorder, GNE myopathy. It is characterized by a slowly progressive defect in proximal and distal skeletal muscles with patients becoming wheel-chair-bound. There are no current approved therapies available for GNE myopathy. ManNAc therapy is currently in advanced clinical trials and has shown signs of slowing the disease progression in a phase 2 trial. The present study aims to understand the effect of GNE mutation on its enzymatic activity and identification of potential small effector molecules. We characterized different GNE mutations (p.Asp207Val, p.Val603Leu, p.Val727Met, p.Ile618Thr and p.Arg193Cys) prevalent in Asian population that were cloned, expressed and purified from Escherichia coli as full-length recombinant proteins. Our study demonstrates that full length GNE can be expressed in E. coli in its active form and analysed for the functional activity. Each mutation showed variation in epimerase and kinase activity and responded to the small effector molecules (metformin, BGP-15 kaempferol, catechin, quercetin) in a differential manner. Our study opens an area for futuristic structural determination of full length GNE and identification of potential therapeutic molecules.

Wavelength-Selective Uncaging of Two Different Photoresponsive Groups on One Effector Molecule for Light-Controlled Activation and Deactivation.

Photocleavable protecting groups (PPGs) play a pivotal role in numerous studies. They enable controlled release of small effector molecules to induce biochemical function. The number of PPGs attached to a variety of effector molecules has grown rapidly in recent years satisfying the high demand for new applications. However, until now molecules carrying PPGs have been designed to activate function only in a single direction, namely the release of the effector molecule. Herein, we present the new approach Two-PPGs-One-Molecule (TPOM) that exploits the orthogonal photolysis of two photoprotecting groups to first release the effector molecule and then to modify it to suppress its induced effect. The moiety resembling the tyrosyl side chain of the translation inhibitor puromycin was synthetically modified to the photosensitive ortho-nitrophenylalanine that cyclizes upon near UV-irradiation to an inactive puromycin cinnoline derivative. Additionally, the modified puromycin analog was protected by the thio-coumarylmethyl group as the second PPG. This TPOM strategy allows an initial wavelength-selective activation followed by a second light-induced deactivation. Both photolysis processes were spectroscopically studied in the UV/vis- and IR-region. In combination with quantum-chemical calculations and time-resolved NMR spectroscopy, the photoproducts of both activation and deactivation steps upon illumination were characterized. We further probed the translation inhibition effect of the new synthesized puromycin analog upon light activation/deactivation in a cell-free GFP translation assay. TPOM as a new method for precise triggering activation/deactivation of effector molecules represents a valuable addition for the control of biological processes with light.

Poly-L Lysine based Approaches for Pretargeted Radioimmunotherapy with Astatine-211

Pretargeted radioimmunotherapy (PRIT) has the potential to increase activity uptake in tumors relative to normal tissue compared with conventional radioimmunotherapy. In pretargeting there is preferably a three step administration of agents with different properties. Firstly the pretargeting molecules (usually antibody conjugates) are administered allowing for maximum tumor targeting during the course of several hours. Secondly, a clearing agent is injected in order to clear unbound pretargeting molecules from the blood circulation. When administering antibody-based pretargeting molecules systemically, they will exhibit a relatively slow clearance from the blood why the clearing agent reduce the absorbed dose to normal tissue and thereby maximize the absorbed dose to the tumors. Thirdly, a radiolabeled effector molecule with high affinity for the pretargeting molecule is administered. The effector is a small molecule that therefore exhibits rapid circulation and blood clearance allowing more of the injected radioactivity to reach the tumor compared with conventional radioimmunotherapy. In addition the small effector molecule also distribute faster within the tumor, rendering a more homogenous activity distribution compared with directly radiolabeled antibodies. Homogenous activity distribution is especially important within Targeted Alpha Therapy where particles ranges in tissue are short (< 100μm). One of the best alpha particle emitters for curative therapies is astatine-211 that have a 7.21 h half-life and one alpha emission per decay. Several different pretargeting approaches exists where two of the most important ones are the biological (strept)Avidine(SA)<->Biotin system and the chemical Tetrazine (Ttz)<>Tetracyclooctane (TCO) system. In either case the pretargeting molecule is functionalized with one of the ingoing components in each system while both the clearing agent and the effector molecule is functionalized with the other. Poly-L Lysine is a versatile scaffold that can be modified in order to function both as effector molecule and as clearing agent in either one of the mentioned pretargeting systems. In this work, different approaches using Poly-L Lysine within pretargeted radioimmunotherapy with astatine-211 are explored. For use as clearing agent, Poly Lysine is functionalized with galactose amine to steer clearance to the liver and succinic anhydride for charge modification. Charge modification is also necessary for use as effector molecule where the poly Lysine in addition is functionalized with a molecule e.g. N-succinimidyl-3(trimethylstannyl)benzoate, in order to allow for incorporation of the radionuclide, astatine211. Poly-L Lysine is also available in different chain-lengths in order to change circulation and clearance properties.

One-step site-specific antibody fragment auto-conjugation using SNAP-tag technology.

Antibody-based diagnostic and therapeutic agents play a substantial role in medicine, especially in cancer management. A variety of chemical, genetic and enzymatic site-specific conjugation methods have been developed for equipping antibodies with effector molecules to generate homogeneous antibody conjugates with tailored properties. However, most of these methods are relatively complicated and expensive and require several reaction steps. Self-labeling proteins such as the SNAP-tag are an innovative solution for addressing these challenges. The SNAP-tag is a modified version of the human DNA repair enzyme alkylguanine-DNA alkyltransferase (AGT), which reacts specifically with O(6)-benzylguanine (BG)-modified molecules via irreversible transfer of an alkyl group to a cysteine residue. It provides a simple, controlled and robust site-specific method for labeling antibodies with different synthetic small effector molecules. Fusing a SNAP-tag to recombinant antibodies allows efficient conjugation of BG-containing substrates by autocatalytic, irreversible transfer of the alkyl group to a cysteine residue in the enzyme's active site under physiological conditions and with a 1:1 stoichiometry. This protocol describes how to generate site-specific SNAP-tag single-chain antibody fragment (scFv) conjugates with different types of BG-modified effector molecules. A specific example is included for the design and production of an scFv-photosensitizer conjugate and its characterization as an immuno-theranostic agent. This protocol includes DNA sequences encoding scFV-SNAP-tag fusion proteins and outlines strategies for expression, purification and testing of the resulting scFv-SNAP-tag-based immuno-conjugates. All experiments can be performed by a graduate-level researcher with basic molecular biology skills within an 8-week time frame.

Poly-L Lysine based Approaches for Pretargeted Radioimmunotherapy with Astatine-211

Pretargeted radioimmunotherapy (PRIT) has the potential to increase activity uptake in tumors relative to normal tissue compared with conventional radioimmunotherapy. In pretargeting there is preferably a three step administration of agents with different properties. Firstly the pretargeting molecules (usually antibody conjugates) are administered allowing for maximum tumor targeting during the course of several hours. Secondly, a clearing agent is injected in order to clear unbound pretargeting molecules from the blood circulation. When administering antibody-based pretargeting molecules systemically, they will exhibit a relatively slow clearance from the blood why the clearing agent reduce the absorbed dose to normal tissue and thereby maximize the absorbed dose to the tumors. Thirdly, a radiolabeled effector molecule with high affinity for the pretargeting molecule is administered. The effector is a small molecule that therefore exhibits rapid circulation and blood clearance allowing more of the injected radioactivity to reach the tumor compared with conventional radioimmunotherapy. In addition the small effector molecule also distribute faster within the tumor, rendering a more homogenous activity distribution compared with directly radiolabeled antibodies. Homogenous activity distribution is especially important within Targeted Alpha Therapy where particles ranges in tissue are short (< 100μm). One of the best alpha particle emitters for curative therapies is astatine-211 that have a 7.21 h half-life and one alpha emission per decay. Several different pretargeting approaches exists where two of the most important ones are the biological (strept)Avidine(SA)<->Biotin system and the chemical Tetrazine (Ttz)<>Tetracyclooctane (TCO) system. In either case the pretargeting molecule is functionalized with one of the ingoing components in each system while both the clearing agent and the effector molecule is functionalized with the other. Poly-L Lysine is a versatile scaffold that can be modified in order to function both as effector molecule and as clearing agent in either one of the mentioned pretargeting systems. In this work, different approaches using Poly-L Lysine within pretargeted radioimmunotherapy with astatine-211 are explored. For use as clearing agent, Poly Lysine is functionalized with galactose amine to steer clearance to the liver and succinic anhydride for charge modification. Charge modification is also necessary for use as effector molecule where the poly Lysine in addition is functionalized with a molecule e.g. N-succinimidyl-3(trimethylstannyl)benzoate, in order to allow for incorporation of the radionuclide, astatine211. Poly-L Lysine is also available in different chain-lengths in order to change circulation and clearance properties.

Bacillus As Potential Probiotics: Status, Concerns, and Future Perspectives.

Spore-forming bacilli are being explored for the production and preservation of food for many centuries. The inherent ability of production of large number of secretory proteins, enzymes, antimicrobial compounds, vitamins, and carotenoids specifies the importance of bacilli in food chain. Additionally, Bacillus spp. are gaining interest in human health related functional food research coupled with their enhanced tolerance and survivability under hostile environment of gastrointestinal tract. Besides, bacilli are more stable during processing and storage of food and pharmaceutical preparations, making them more suitable candidate for health promoting formulations. Further, Bacillus strains also possess biotherapeutic potential which is connected with their ability to interact with the internal milieu of the host by producing variety of antimicrobial peptides and small extracellular effector molecules. Nonetheless, with proposed scientific evidences, commercial probiotic supplements, and functional foods comprising of Bacillus spp. had not gained much credential in general population, since the debate over probiotic vs pathogen tag of Bacillus in the research and production terrains is confusing consumers. Hence, it’s important to clearly understand the phenotypic and genotypic characteristics of selective beneficial Bacillus spp. and their substantiation with those having GRAS status, to reach a consensus over the same. This review highlights the probiotic candidature of spore forming Bacillus spp. and presents an overview of the proposed health benefits, including application in food and pharmaceutical industry. Moreover, the growing need to evaluate the safety of individual Bacillus strains as well as species on a case by case basis and necessity of more profound analysis for the selection and identification of Bacillus probiotic candidates are also taken into consideration.

Light-controlled gene expression in yeast using photocaged Cu2+

The manipulation of cellular function, such as the regulation of gene expression, is of great interest to many biotechnological applications and often achieved by the addition of small effector molecules. By combining effector molecules with photolabile protecting groups that mask their biological activity until they are activated by light, precise, yet minimally invasive, photocontrol is enabled. However, applications of this trendsetting technology are limited by the small number of established caged compound-based expression systems. Supported by computational chemistry, we used the versatile photolabile chelator DMNP-EDTA, long-established in neurobiology for photolytic Ca2+ release, to control Cu2+ release upon specific UV-A irradiation. This permits light-mediated control over the widely used Cu2+-inducible pCUP1 promoter from S. cerevisiae and thus constitutes the first example of a caged metal ion to regulate recombinant gene expression. We screened our novel DMNP-EDTA-Cu system for best induction time and expression level of eYFP with a high-throughput online monitoring system equipped with an LED array for individual illumination of every single well. Thereby, we realized a minimally invasive, easy-to-control, parallel and automated optical expression regulation via caged Cu2+ allowing temporal and quantitative control as a beneficial alternative to conventional induction via pipetting CuCl2 as effector molecule.

How Does Host Carbon Concentration Modulate the Lifestyle of Postharvest Pathogens during Colonization?

Postharvest pathogens can penetrate fruit by breaching the cuticle or directly through wounds, and they show disease symptoms only long after infection. During ripening and senescence, the fruit undergo physiological processes accompanied by a decline in antifungal compounds, which allows the pathogen to activate a mechanism of secretion of small effector molecules that modulate host environmental pH. These result in the activation of genes under their optimal pH conditions, enabling the fungus to use a specific group of pathogenicity factors at each particular pH. New research suggests that carbon availability in the environment is a key factor triggering the production and secretion of small pH-modulating molecules: ammonia and organic acids. Ammonia is secreted under limited carbon and gluconic acid under excess carbon. This mini review describes our most recent knowledge of the mechanism of activation of pH-secreted molecules and their contribution to colonization by postharvest pathogens to facilitate the transition from quiescence to necrotrophic lifestyle.

Use of bacteriophage to target bacterial surface structures required for virulence: a systematic search for antibiotic alternatives.

Bacteriophages (phage) that infect pathogenic bacteria often attach to surface receptors that are coincidentally required for virulence. Receptor loss or modification through mutation renders mutants both attenuated and phage resistant. Such attenuated mutants frequently have no apparent laboratory growth defects, but in the host, they fail to exhibit properties needed to produce disease such as mucosal colonization or survival within professional phagocytic cells. The connection between attenuation and phage resistance has been exploited in experimental demonstrations of phage therapy. In such experiments, phage resistant mutants that arise naturally during therapy are inconsequential because of their attenuated status. A more contemporary approach to exploiting this connection involves identifying small effector molecules, identified in high-throughput screens, that inhibit one or more of the steps needed to produce a functioning phage receptor. Since such biosynthetic steps are unique to bacteria, inhibitors can be utilized therapeutically, in lieu of antibiotics. Also, since the inhibitor is specific to a particular bacterium or group of bacteria, no off-target resistance is generated in the host's commensal bacterial population. This brief review covers examples of how mutations that confer phage resistance produce attenuation, and how this coincidental relationship can be exploited in the search for the next generation of therapeutic agents for bacterial diseases.

A Comparative Study on Techniques for the Determination of Permeabilization Events on Charged and Uncharged Lipid Bilayers

Host defense peptides (HDPs) are small effector molecules of the innate immune system building up the first defense barrier when it comes to infections. Infectious diseases are commonly treated with antibiotics leading to an increase in antibiotic resistances among clinically relevant pathogens. Therefore, understanding the mode of function of alternative agents with antibiotic function is undeniable for the successful development of new antibiotics opening new treatment options. To understand recognition-, recruiting- and permeabilization-processes on membrane surfaces a set of structurally diverse peptides is investigated regarding their membrane active function on charged and uncharged model membranes. Lipid bilayers are reconstituted from pure DOPC to mimic uncharged membranes and from POPE/POPG in a ratio of 4:1 to mimic the inner leaflet of the bactericidal cell envelope with an overall negative surface charge. Permeabilization events induced by the antimicrobial peptides LL32, Arenicin-1 and hBD3-l as well as the ionophore Nonactin and α-Hemolysin of Staphylococcus aureus are investigated with respect to the membrane systems stated above, buffer composition (high-salt vs. low-salt) and temperature conditions. A combination of a complex array of biophysical techniques is taken into account for studying permeabilization events with nearly no limitations - MONTAL-MUELLER-Setup for planar lipid bilayers, tethered membrane technology for supported bilayers, liposome-based assays (Calcein release assay, potassium iodide quenching, FRET-intercalation assay) and a chip-based pore spanning dye release assay. These methods allow to investigate the whole spectrum ranging from single pore characterization to the statistical analysis of either large membrane areas or a huge number of single membranes.

Subacute cutaneous lupus erythematosus induced by lansoprazole

A 48-year-old woman presented us with multiple annular erythematous-squamous plaques over trunk and limbs appeared 3 weeks before. There was no mucosal involvement. Her medical history included systemic lupus erythematosus (LES), diagnosed 3 years before and currently no longer treated, and gastroesophageal reflux for which she was in therapy with lansoprazole (30mg/daily) from 2months.Microscopy in 10 % potassium hydroxide and culture for mycoses was negative. Routine laboratory investigations were normal, antinuclear antibodies (ANA) negative, and anti-Ro/SSA antibodies positive (titer 1/80). Histology of an annular lesion showed atrophy of the epidermis, hydropic degeneration of the basal layer, edema in the upper dermis, and perivascular and periadnexial lymphohistiocytic infiltrate, consistent with subacute cutaneous lupus erythematosus (SCLE). A direct immunofluorescence test was negative. The patient received oral methylprednisolone (0.5 mg/kg/daily), topical steroids, and continued lansoprazolewith improvement but without complete resolution of the lesions. Therefore, lansoprazole was suspected to be responsible of the eruption and replaced by alginate and bicarbonate sodium with complete remission in a few days. Hence, diagnosis of drug-induced SCLE (DI-SCLE) was made. SCLE is a subtype of cutaneous lupus erythematosus that usually manifests as annular, psoriasiform lesions with limited systemic involvement and typically associated with positive ANA, anti-Ro/SSA, and anti-La/SSB antibodies. It may also be induced or exacerbated by drugs, including thiazide diuretics, calcium channel blockers, and also proton pump inhibitors (PPI) [1]. DI-SCLE does not differ clinically, histopathologically, or immunologically from idiopathic SCLE even though some authors have emphasized that DI-SCLE presented more disseminated cutaneous manifestations (as in our patient) and more frequent occurrence of malar rash and bullous, targetoid, and vasculitic lesions [1]. Patients with autoimmune diseases are more prone to druginduced or exacerbated SCLE [1], but the pathogenesis of DISCLE is not completely understood. However, it may be correlated to antimicrobial peptides (AMPs), small effector molecules of the innate immune system with well-known antimicrobial activity [2, 3]. In fact, several AMPs are increased in CLE at both gene and protein levels. In particular, AMPs were found to be significantly more highly induced in subacute CLE as compared with discoid LE and LE tumidus [2]. This might also explain the low prevalence of skin infections in CLE [2]. The selectivity of AMPs to bacterial cells relies on their cationic structures that are crucial for the interaction with negatively charged bacterial membranes [3]. Cathelicidins are a family of evolutionarily conserved AMPs, and hCAP-18 is the only cathelicidin in humans. Proteolytic cleavage of this preprotein releases a 37-residue known as LL-37 that is secreted by bone marrow cells, circulating leukocytes, and numerous types of epithelial tissues, such as skin. LL-37 could mediate innate immunity through regulating chemotaxis of leukocytes and production of cytokines at sites of infection/ inflammation as well as promoting re-epithelization during wound healing. LL-37 orients near the surface of phospholipid bilayers and forms oligomeric structures possessing the ability to disrupt cell membrane [3]. Depending on cellular context, autophagy may serve as a pro-death either pro-survival mechanism through p53 activation [3]. Human LL-37 and its derivatives may contribute to the control of immune-mediated inflammatory diseases through the regulation of proand antiinflammatory cytokines [4]. * Sanja Javor javor.med@gmail.com

Solution structure and functional studies of the highly potent equine antimicrobial peptide DEFA1

Defensins are small effector molecules of the innate immune system that are present in almost all organisms including plants and animals. These peptides possess antimicrobial activity against a broad range of microbes including bacteria, fungi and viruses and act as endogenous antibiotics. α-Defensins are a subfamily of the defensin family and their expression is limited to specific tissues. Equine DEFA1 is an enteric α-defensin exclusively secreted by Paneth cells and shows an activity against a broad spectrum of microbes, including typical pathogens of the horse such as Rhodococcus equi, various streptococci strains, Salmonella choleraesuis, and Pasteurella multocida. Here, we report the three-dimensional structure of DEFA1 solved by NMR-spectroscopy and demonstrate its specific function of aggregating various phospholipids.

The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity.

The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.

Hysteresis in human UDP-glucose dehydrogenase is due to a restrained hexameric structure that favors feedback inhibition.

Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.

Small RNA-Mediated Epigenetic Myostatin Silencing

Myostatin (Mstn) is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as Duchenne muscular dystrophy. Here we describe a novel Mstn blockade approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in two differentiated mouse muscle cell lines. Silencing is sensitive to treatment with the histone deacetylase inhibitor trichostatin A, and the silent state chromatin mark H3K9me2 is enriched at the Mstn promoter following siRNA transfection, suggesting epigenetic remodeling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for Mstn and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders.

Efficient Synthesis and Applications of Peptides containing Adenylylated Tyrosine Residues

During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.

Expression of antimicrobial peptides in different subtypes of cutaneous lupus erythematosus

Antimicrobial peptides (AMPs) are small effector molecules of the innate immune system with well-known antimicrobial activity. Skin infections rarely occur in patients with cutaneous lupus erythematosus (CLE), and AMP expression in CLE has not been previously evaluated. We aimed to determine the expression of several important AMPs in 3 different subtypes ofCLE. Skin lesions were analyzed for the gene and protein expression of human β-defensin (hBD)-1, -2, and -3; RNase-7; the cathelicidin LL-37; and psoriasin (S100A7) using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Skin biopsy specimens of 96 study participants including 47 patients with CLE (15 patients with discoid lupus erythematosus [LE], 11 patients with subacute CLE, and 21 patients with LE tumidus), 34 patients with psoriasis, and 15 healthy control subjects were evaluated in this study. HBD-2, hBD-3, LL-37, and psoriasin were significantly more highly expressed in CLE as compared with healthy controls, and most AMPs were significantly more highly induced in subacute CLE as compared with discoid LE and LE tumidus. AMP gene expression paralleled well with AMP protein expression in CLE and controls. Subacute CLE and discoid LE showed a similar correlation of AMP gene expression (significant correlations between hBD-1 and RNase-7, hBD-2 and hBD-3, hBD-2 and psoriasin, and hBD-3 and psoriasin). The relatively small number of samples and the lack of analysis of the lesional bacterial colonization are a limitation. Several AMPs are increased in CLE at both gene and protein levels. This could explain the low prevalence of skin infections in CLE. It remains to be elucidated whether AMPs play a pathogenic role in CLE.

Effectors, effectors et toujours des effectors

Effectors, effectors <i>et toujours des</i> effectors