Metabolically-incorporated deuterium in myelin localized by neutron diffraction and identified by mass spectrometry.

Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially with respect to its biosynthesis and assembly during its normal formation, maintenance, and pathological breakdown. To explore the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of in vivo labeling by metabolically incorporating non-toxic levels of deuterium (2H; D) via drinking water into a pregnant dam (D-dam) and her developing embryos. All of the mice were sacrificed when the pups (D-pups) were 55 days old. Myelinated sciatic nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed (trigeminal nerves) were frozen for mass spectrometry (MS). The diffraction patterns of the nerves from deuterium-fed mice (D-mice) vs. the controls (H-mice) had major differences in the intensities of the Bragg peaks but no appreciable differences in myelin periodicity. Neutron scattering density profiles showed an appreciable increase in density at the center of the lipid-rich membrane bilayer. This increase was greater in D-pups than in D-dam, and its localization was consistent with deuteration of lipid hydrocarbon, which predominates over transmembrane protein in myelin. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that in the pups the percentage of lipids that had one or more deuterium atoms was uniformly high across lipid species (97.6% â€‹± â€‹2.0%), whereas in the mother the lipids were substantially less deuterated (60.6% â€‹± â€‹26.4%) with levels varying among lipid species and subspecies. The mass distribution pattern of deuterium-containing isotopologues indicated the fraction (in %) of each lipid (sub-)species having one or more deuteriums incorporated: in the D-pups, the pattern was always bell-shaped, and the average number of D atoms ranged from a low of ∼4 in fatty acid to a high of ∼9 in cerebroside. By contrast, in D-dam most lipids had more complex, overlapping distributions that were weighted toward a lower average number of deuteriums, which ranged from a low of ∼3-4 in fatty acid and in one species of sulfatide to a high of 6-7 in cerebroside and sphingomyelin. The consistently high level of deuteration in D-pups can be attributed to their de novo lipogenesis during gestation and rapid, postnatal myelination. The widely varying levels of deuteration in D-dam, by contrast, likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understanding of the de novo synthesis and turnover of specific lipids in normal and abnormal myelin, our results also suggest application to studies on myelin proteins (which constitute only 20-30% by dry mass of the myelin, vs. 70-80% for lipid), as well as more broadly to the molecular constituents of other biological tissues.

Hepatocellular carcinoma: French Intergroup Clinical Practice Guidelines for diagnosis, treatment and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO, AFEF, SIAD, SFR/FRI).

INTRODUCTION: This document is a summary of the French Intergroup guidelines regarding the management of hepatocellular carcinoma (HCC) published in March 2019. METHOD: It is a collaborative work under the auspices of most of the French medical societies involved in the management of HCC. It is based on the previous guidelines published in 2017. Recommendations are graded in 3 categories according to the level of evidence of data found in the literature. RESULTS: The diagnosis and staging of HCC is essentially based on clinical, biological and imaging features. A pathological analysis obtained by a biopsy of tumoral and non-tumoral liver is recommended. HCCs can be divided into 2 groups, taking into account not only the tumor stage, but also liver function. HCCs accessible to curative treatments are tumors that are in Milan criteria or with an AFP score ≤ 2, mainly treated by surgical resection, local ablation or liver transplantation. Intermediate and advanced HCCs with no liver insufficiency, accessible only to palliative treatments, benefit from TACE, SIRT or systemic therapy according to the presence or absence of macrovascular invasion or extrahepatic spread. CONCLUSION: Such recommendations are in permanent optimization and each individual case must be discussed in a multidisciplinary expert board.

Functional metagenomics of the thioredoxin superfamily.

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

GADL1 is a multifunctional decarboxylase with tissue-specific roles in ß-alanine and carnosine production.

Carnosine and related ß-alanine-containing peptides are believed to be important antioxidants, pH buffers, and neuromodulators. However, their biosynthetic routes and therapeutic potential are still being debated. This study describes the first animal model lacking the enzyme glutamic acid decarboxylase-like 1 (GADL1). We show that Gadl1-/- mice are deficient in ß-alanine, carnosine, and anserine, particularly in the olfactory bulb, cerebral cortex, and skeletal muscle. Gadl1-/- mice also exhibited decreased anxiety, increased levels of oxidative stress markers, alterations in energy and lipid metabolism, and age-related changes. Examination of the GADL1 active site indicated that the enzyme may have multiple physiological substrates, including aspartate and cysteine sulfinic acid. Human genetic studies show strong associations of the GADL1 locus with plasma levels of carnosine, subjective well-being, and muscle strength. Together, this shows the multifaceted and organ-specific roles of carnosine peptides and establishes Gadl1 knockout mice as a versatile model to explore carnosine biology and its therapeutic potential.

Curative Irradiation Treatment of Hepatocellular Carcinoma: A Multicenter Phase 2 Trial.

PURPOSE: Liver transplantation is the standard definitive treatment for nonmetastatic hepatocellular carcinoma (HCC). However, less than 5% of patients are ultimately candidates as a result of frequent comorbidities and graft shortage. The aim of this study was to evaluate stereotactic body radiation therapy (SBRT) as an ablative treatment for inoperable HCC. METHODS AND MATERIALS: A prospective phase 2 trial included newly diagnosed single HCC lesions that were without extrahepatic extension and that were deemed unsuitable for standard locoregional therapies, with a tumor size ranging from 1 to 6 cm. The SBRT dose was 45 Gy in 3 fractions. Primary endpoint was the local control of irradiated HCC at 18 months, defined by Response Evaluation Criteria in Solid Tumors. RESULTS: Forty-three patients were treated and evaluable. Median follow-up was 4.0 years (range, 1.2-4.6 years). All 43 patients had cirrhosis; 37 (88%) were Child-Pugh grade A and 5 (12%) grade B (1 missing data). No patients had received prior local treatment. Thirteen patients (31%) presented grade ≥3 acute adverse events, including 8 patients with an abnormality of the liver function tests (19%). Three patients (10%) experienced a decline in Child-Pugh at 3 months post-SBRT. The 18-month local control rate was 98% (95% confidence interval, 85%-99%). The 18-month overall survival rate was 72% (range, 56%-83%). Median overall survival was 3.5 years. CONCLUSIONS: Local control and overall survival after SBRT for untreated solitary HCC were excellent despite candidates being unfit for transplantation, resection, ablation, or embolization treatments. SBRT should be considered as a bridge to transplant or as definitive therapy for those ineligible for transplant.

Molecular structure and function of myelin protein P0 in membrane stacking.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.

Gastric cancer: French intergroup clinical practice guidelines for diagnosis, treatments and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO).

INTRODUCTION: This document is a summary of the French Intergroup guidelines regarding the management of gastric cancer published in October 2016, available on the website of the French Society of Gastroenterology (SNFGE) (www.tncd.org), updated in October 2017. METHODS: This collaborative work was realized under the auspices of several French medical societies involved in management of gastric cancer. Recommendations are graded in three categories (A-C), according to the amount of evidence found in the literature until July 2017. RESULTS: There are several known risk factors for gastric cancer, including Helicobacter pylori and genetic predispositions, both requiring a specific screening for patients and their relatives. The diagnosis and staging evaluation are essentially based on gastroscopy plus biopsies and computed tomography scan. The endoscopic ultrasonography can be used for superficial tumors in case of discussion for endoscopic resection (T1N0). For local disease (N+ and/or T > T1), the strategic therapy is based on surgery associated with perioperative chemotherapy. In the absence of preoperative treatment (for any raison), the postoperative chemoradiotherapy (or chemotherapy) should be discussed for patients with stage II or III tumor. For metastatic disease, the treatment is based on "palliative" chemotherapy consisting in a doublet or triplet regimens depending of age, performance status and HER2 tumor status. For patients with limited metastatic disease, surgical resection could be discussed in multidisciplinary meeting in case of stable disease after chemotherapy. CONCLUSION: These guidelines in gastric cancer are done to help decision for daily clinical practice. These recommendations are permanently being reviewed. Each individual case must be discussed within a multidisciplinary team.

Publisher Correction: Molecular mechanisms of Charcot-Marie-Tooth neuropathy linked to mutations in human myelin protein P2.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Molecular mechanisms of Charcot-Marie-Tooth neuropathy linked to mutations in human myelin protein P2.

Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the ß barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.

Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line.

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions.

Intensity-modulated radiation therapy from 70Gy to 80Gy in prostate cancer: six- year outcomes and predictors of late toxicity.

OBJECTIVE: To report grade ≥2 overall late rectal and urinary toxicities in patients (pts) with prostate cancer treated by intensity-modulated radiotherapy (IMRT) at 3 dose-levels. Identify predictors of radiation toxicity and report biochemical progression free survival (bPFS). METHODS: A total of 277 pts were treated with 70Gy (10.8%), 74Gy (63.9%) and 80 Gy (25.3%) using IMRT without pelvic irradiation were analyzed. Short or long-course androgen deprivation therapy (ADT) was allowed in 46.1% of pts. The toxicity was described using the Common Terminology Criteria for Adverse Events (CTCAE) v4.0 scale. Cox regression models addressed demographics, disease and dosimetry characteristics as potential predictors of late grade ≥2 toxicity after adjusting for other modifying factors. RESULTS: The median follow-up was 77 months (range 15; 150). There was no grade ≥4 toxicity. The 5-year cumulative rate of grade ≥2 late rectal and urinary toxicities was 6.3% (95% CI = 3.8%; 10.3%) and 25.3% (95% CI = 19.8%; 31.8%) respectively. In multivariate analysis, only the dose (80Gy vs 74 and 70Gy) was found to increase the risk of rectal toxicity (HR = 2.96 [1.07; 8.20]). For pts receiving 74 Gy, International Prostate Symptom Score (IPSS) at baseline ≥8 (HR = 2.40 [1.08; 5.35]) and dose ≥73Gy delivered in more than 2% of bladder (D2%) were found to be predictors of bladder toxicity (HR = 3.29 [1.36; 7.98]). The 5-year biochemical relapse free survival was 81.0% [74.5%; 86.0%] in the entire population, 97.5% [83.5%; 99.6%] in the low risk group, 84.9% [76.7%; 90.3%] in the intermediate risk group and 66.4% [51.8%; 77.4%] in the high-risk group. D'Amico low (HR = 0.09 [0.01; 0.69]) and intermediate risk groups (HR = 0.50 [0.28; 0.88]) as well as PSA nadir ≥0.2 ng/ml (HR = 1.79 [1.01; 3.21]) were predictive of biochemical relapse. CONCLUSIONS: The rate of late rectal toxicity increased with higher doses, while Dmax ≥74Gy, D2% ≥ 73Gy for bladder wall and baseline IPSS ≥8 increased late urinary toxicity.

Collapsin response mediator protein 2: high-resolution crystal structure sheds light on small-molecule binding, post-translational modifications, and conformational flexibility.

Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands.

Tyrosine Hydroxylase Binding to Phospholipid Membranes Prompts Its Amyloid Aggregation and Compromises Bilayer Integrity.

Tyrosine hydroxylase (TH), a rate-limiting enzyme in the synthesis of catecholamine neurotransmitters and hormones, binds to negatively charged phospholipid membranes. Binding to both large and giant unilamellar vesicles causes membrane permeabilization, as observed by efflux and influx of fluorescence dyes. Whereas the initial protein-membrane interaction involves the N-terminal tail that constitutes an extension of the regulatory ACT-domain, prolonged membrane binding induces misfolding and self-oligomerization of TH over time as shown by circular dichroism and Thioflavin T fluorescence. The gradual amyloid-like aggregation likely occurs through cross-ß interactions involving aggregation-prone motives in the catalytic domains, consistent with the formation of chain and ring-like protofilaments observed by atomic force microscopy in monolayer-bound TH. PC12 cells treated with the neurotoxin 6-hydroxydopamine displayed increased TH levels in the mitochondrial fraction, while incubation of isolated mitochondria with TH led to a decrease in the mitochondrial membrane potential. Furthermore, cell-substrate impedance and viability assays showed that supplementing the culture media with TH compromises cell viability over time. Our results revealed that the disruptive effect of TH on cell membranes may be a cytotoxic and pathogenic factor if the regulation and intracellular stability of TH is compromised.

Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners.

Decisions about Limiting Treatment in Cancer Patients: A Systematic Review and Clinical Ethical Analysis of Reported Variables.

BACKGROUND: Survey research indicates that decisions about the provision and limitation of treatment near the end of life in patients with cancer vary considerably. OBJECTIVES: The study objectives were to review the evidence on variables associated with explicit decisions about limitation of treatment in patients with cancer and to critically appraise the factors from a clinical ethics perspective. METHODS: A search was conducted of studies published in EMBASE, CINAHL, PsycINFO, Assia, Current Contents Medicine, Belit, and Euroethics before February 5, 2014. Eligible studies reported data on explicit treatment limitation in patients with cancer and included a statistical analysis on possibly associated factors. Information on study participants, types of limited treatment, and variables associated with limiting treatment were extracted by two researchers independently. Data synthesis was performed jointly by researchers from oncology, medical ethics, and social sciences. RESULTS: The search yielded 897 publications, of which 7 were relevant for this review. Factors significantly associated with decisions about limitation of treatment could be distinguished in three categories: first, sociodemographic variables such as the ethnic background of patients; second, health- or treatment-related variables including a lack of capacity in patients with cancer; and third, patients' preferences and the role of relatives in decisions about limitation of treatment. Limitations to this study are that the studies lacked a predefined hypothesis and they all had been conducted in Western countries. CONCLUSION: The identified variables raise ethical issues with regards to possible influence of value judgments underlying decisions about limitation of treatment in end-of-life care.

Stereotactic body radiation therapy as an ablative treatment for inoperable hepatocellular carcinoma.

PURPOSE: To describe efficacy and safety of stereotactic body radiation therapy (SBRT) for the treatment of inoperable hepatocellular carcinoma. METHODS: The records of 77 consecutive patients treated with SBRT for 97 liver-confined HCC were reviewed. A total dose of 45Gy in 3 fractions was prescribed to the 80% isodose line. Local control (LC), overall survival (OS), progression-free survival (PFS) and toxicity were studied. RESULTS: The median follow-up was 12months. The median tumor diameter was 2.4cm. The LC rate was 99% at 1 and 2years. The 1 and 2-year OS were 81.8% and 56.6% respectively. The median time to progression was 9months (0-38). The rate of hepatic toxicity was 7.7% [1.6-13.7], 14.9% [5.7-23.2] and 23.1% [9.9-34.3] at 6months, 1year and 2years respectively. In multivariate analysis, female gender (HR 7.87 [3.14-19.69]), a BCLC B-C stage (HR 3.71 [1.41-9.76]), a sum of all lesion diameters ⩾2cm (HR 7.48 [2.09-26.83]) and a previous treatment (HR 0.10 [0.01-0.79]) were independent prognostic factors of overall survival. CONCLUSION: SBRT allows high local control for inoperable hepatocellular carcinomas. It should be considered when an ablative treatment is indicated in Child A patients.

Arc is a flexible modular protein capable of reversible self-oligomerization.

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.

Identification of a novel lytic peptide for the treatment of solid tumours.

Originally known as host defence peptides for their substantial bacteriotoxic effects, many cationic antimicrobial peptides also exhibit a potent cytotoxic activity against cancer cells. Their mode of action is characterized mostly by electrostatic interactions with the plasma membrane, leading to membrane disruption and rapid necrotic cell death. In this work, we have designed a novel cationic peptide of 27 amino acids (Cypep-1), which shows efficacy against a number of cancer cell types, both in vitro and in vivo, while normal human fibroblasts were significantly less affected. Surface plasmon resonance experiments as well as liposome leakage assays monitored by fluorescence spectroscopy revealed a substantial binding affinity of Cypep-1 to negatively charged liposomes and induced significant leakage of liposome content after exposure to the peptide. The observed membranolytic effect of Cypep-1 was confirmed by scanning electron microscopy (SEM) as well as by time-lapse confocal microscopy. Pharmacokinetic profiling of Cypep-1 in rats showed a short plasma half-life after i.v. injection, followed mainly by retention in the liver, spleen and kidneys. Extremely low concentrations within the organs of the central nervous system indicated that Cypep-1 did not pass the blood-brain-barrier. Local treatment of 4T1 murine mammary carcinoma allografts by means of a single local bolus injection of Cypep-1 led to a significant reduction of tumour growth in the following weeks and prolonged survival. Detailed histological analysis of the treated tumours revealed large areas of necrosis. In sum, our findings show that the novel cationic peptide Cypep-1 displays a strong cytolytic activity against cancer cells both in vitro and in vivo and thus holds a substantial therapeutic potential.

The N-terminal sequence of tyrosine hydroxylase is a conformationally versatile motif that binds 14-3-3 proteins and membranes.

Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the synthesis of catecholamine neurotransmitters, and a reduction in TH activity is associated with several neurological diseases. Human TH is regulated, among other mechanisms, by Ser19-phosphorylation-dependent interaction with 14-3-3 proteins. The N-terminal sequence (residues 1-43), which corresponds to an extension to the TH regulatory domain, also interacts with negatively charged membranes. By using X-ray crystallography together with molecular dynamics simulations and structural bioinformatics analysis, we have probed the conformations of the Ser19-phosphorylated N-terminal peptide [THp-(1-43)] bound to 14-3-3γ, free in solution and bound to a phospholipid bilayer, and of the unphosphorylated peptide TH-(1-43) both free and bilayer bound. As seen in the crystal structure of THp-(1-43) complexed with 14-3-3γ, the region surrounding pSer19 adopts an extended conformation in the bound state, whereas THp-(1-43) adopts a bent conformation when free in solution, with higher content of secondary structure and higher number of internal hydrogen bonds. TH-(1-43) in solution presents the highest mobility and least defined structure of all forms studied, and it shows an energetically more favorable interaction with membranes relative to THp-(1-43). Cationic residues, notably Arg15 and Arg16, which are the recognition sites of the kinases phosphorylating at Ser19, are also contributing to the interaction with the membrane. Our results reveal the structural flexibility of this region of TH, in accordance with the functional versatility and conformational adaptation to different partners. Furthermore, this structural information has potential relevance for the development of therapeutics for neurodegenerative disorders, through modulation of TH-partner interactions.