Membrane remodelling by a lipidated endosomal sorting complex required for transport-III chimera, in vitro.

The complexity of eukaryotic cells is underscored by the compartmentalization of chemical signals by phospholipid membranes. A grand challenge of synthetic biology is building life from the 'bottom-up', for the purpose of generating systems simple enough to precisely interrogate biological pathways or for adapting biology to perform entirely novel functions. Achieving compartmentalization of chemistries in an addressable manner is a task exquisitely refined by nature and embodied in a unique membrane remodelling machinery that pushes membranes away from the cytosol, the ESCRT-III (endosomal sorting complex required for transport-III) complex. Here, we show efforts to engineer a single ESCRT-III protein merging functional features from its different components. The activity of such a designed ESCRT-III is shown by its ability to drive the formation of compartments encapsulating fluorescent cargo. It appears that the modular nature of ESCRT-III allows its functional repurposing into a minimal machinery that performs sophisticated membrane remodelling, therefore enabling its use to create eukaryotic-like multi-compartment architectures.

TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity.

The vgf gene has been identified as an energy homeostasis regulator. Vgf encodes a 617-aa precursor protein that is processed to yield an incompletely characterized panel of neuropeptides. Until now, it was an unproved assumption that VGF-derived peptides could regulate metabolism. Here, a VGF peptide designated TLQP-21 was identified in rat brain extracts by means of immunoprecipitation, microcapillary liquid chromatography-tandem MS, and database searching algorithms. Chronic intracerebroventricular (i.c.v.) injection of TLQP-21 (15 mug/day for 14 days) increased resting energy expenditure (EE) and rectal temperature in mice. These effects were paralleled by increased epinephrine and up-regulation of brown adipose tissue beta2-AR (beta2 adrenergic receptor) and white adipose tissue (WAT) PPAR-delta (peroxisome proliferator-activated receptor delta), beta3-AR, and UCP1 (uncoupling protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic and anorexigenic neuropeptides was unchanged. Furthermore, in mice that were fed a high-fat diet for 14 days, TLQP-21 prevented the increase in body and WAT weight as well as hormonal changes that are associated with a high-fat regimen. Biochemical and molecular analyses suggest that TLQP-21 exerts its effects by stimulating autonomic activation of adrenal medulla and adipose tissues. In conclusion, we present here the identification in the CNS of a previously uncharacterized VGF-derived peptide and prove that its chronic i.c.v. infusion effected an increase in EE and limited the early phase of diet-induced obesity.

Chronic administration of olanzapine induces metabolic and food intake alterations: a mouse model of the atypical antipsychotic-associated adverse effects.

RATIONALE: Most of atypical antipsychotics (AAPs) are highly related to a major risk of metabolic drawbacks leading to dyslipidemia and obesity. OBJECTIVE: To set up a mouse model of the AAP-associated weight gain in mice under the influence of chronic olanzapine regimen. MATERIALS AND METHODS: Female mice were housed in pairs and habituated to spontaneous feeding with a high-palatable diet (10% sucrose wet mash). Firstly, we orally administered olanzapine (0.75, 1.5 and 3 mg/kg), evaluating body weight and periuterine fat mass, as well as insulin, non-esterified fatty acids, triglycerides, and glucose levels. In a second experiment, we assessed the effect of olanzapine on energy expenditure through indirect calorimetry (IC). A third experiment was conducted to investigate the effects of olanzapine on a high fat-high sweet palatable diet (10% sucrose + 30% fat, HF-HS) in mice implanted with subcutaneous osmotic mini-pumps. Locomotor activity was also assessed. RESULTS: In experiment 1, the highest dose of chronically administered olanzapine (3 mg/kg) induced significant weight gain accompanied by augmentation of periuterine fat depots, with no changes in locomotor activity. In experiment 2, chronic administration did not alter energy expenditure, whereas, decreased respiratory quotient (RQ). In experiment 3, subcutaneously infused olanzapine evidenced a dose and time-dependent increase of body weight and HF-HS diet consumed. Notably, serum analyses revealed a hyperinsulinemia together with increased levels of triglycerides and glucose. CONCLUSIONS: In this study, we describe in female mice metabolic alterations matching the metabolic syndrome, thus resembling the clinical situation of schizophrenic patients taking AAPs.

Loss of ubiquitin binding is a unifying mechanism by which mutations of SQSTM1 cause Paget's disease of bone.

Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.

Psychosocial stress affects energy balance in mice: modulation by social status.

Stress has been associated with changes in eating behaviour and food preferences. Moreover, psychosocial and socio-economical challenges have been related with neuroendocrine-autonomic dysregulation followed by visceral obesity and associated risk factors for disease. In the current study, we provide a model of body weight development, food intake, energy expenditure of subordinate and dominant mice under psychosocial stress either in the presence of a standard diet or of a high palatable diet. When only standard chow was available stressed animals consumed more food in comparison to the control counterpart. Moreover, subordinate mice, at the end of the stress period were heavier in comparison to dominant animals. This last result was due to a decrease in the caloric efficiency of dominant animals in comparison to subordinates. Confirming this, the results of the experiment 2 showed that dominant mice significantly increase their energy expenditure at the end of the chronic psychosocial stress procedure in comparison to subordinate mice, as measured by indirect calorimetry. When a palatable high fat diet was available subordinate animals became heavier in comparison with both dominant and control animals. No differences in the caloric intake were found between groups. Subordinate mice ingested more calories from fat than controls, while dominant animals ingested more calories from carbohydrates. These results suggest that psychosocial stress can be a risk factor for overeating and weight gain in mice. However, social status influences the extent to which an individual keeps up with adverse environment, influencing the vulnerability toward stress related disorders.

Structural and functional studies of mutations affecting the UBA domain of SQSTM1 (p62) which cause Paget's disease of bone.

Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Paget's disease of bone. The missense mutations resolve into those which retain [P392L (Pro(392)-->Leu), G411S] or abolish (M404V, G425R) the ability of the isolated UBA domain to bind Lys-48-linked polyubiquitin. These effects can be rationalized with reference to the solution structure of the UBA domain, which we have determined by NMR spectroscopy. The UBA domain forms a characteristic compact three-helix bundle, with a hydrophobic patch equivalent to that previously implicated in ubiquitin binding by other UBA domains. None of the mutations affect overall folding of the UBA domain, but both M404V and G425R involve residues in the hydrophobic patch, whereas Pro-392 and Gly-411 are more remote. A simple model assuming the isolated UBA domain is functioning as a compact monomer can explain the effects of the mutations on polyubiquitin binding. The P392L and G411S mutations do however have subtle local effects on secondary structure, which may become more relevant in full-length SQSTM1. Identification of the in vivo ubiquitylated substrates of SQSTM1 will be most informative in determining the functional significance of the SQSTM1-ubiquitin interaction, and consequences of the disease-associated mutations.

Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein.

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including SPR3 in certain specialized epithelia normally subjected to mechanical trauma. We have expressed recombinant human SPR3 in order to study its cross-linking properties. It serves as a complete substrate for, and is cross-linked at similar efficiencies by, the three enzymes (TGases 1, 2 and 3) that are widely expressed in many epithelia. Multiple adjacent glutamines (4, 5, 16, 17, 18, 19 and 167) and lysines (6, 21, 164, 166 and 168) of only head and tail domain sequences are used for cross-linking. However, each enzyme preferentially uses certain residues on the head domain. Moreover, our in vitro data suggest a defined temporal order of cross-linking of SPR3 in vivo: It is first cross-linked by TGase 3 into short intra- and inter-chain oligomers which are later further cross-linked to the CE by TGase 1. To investigate the absence of cross-linking in the central domain (e.g. lysine in position 2 of each of the 16 repeats) we performed structural studies on recombinant SPR3 and on a synthetic peptide containing three repeats of the central domain. 2D H-1 NMR spectroscopy, TOCSY and ROESY, shows strong and medium intensity NOEs connectivities along the amino acid sequence with one weak long range NOE contact between Thr and Cys of subsequent repeats. Distance geometry computation on the basis of intensities of NOEs found generated 50 compatible structures grouped in three main families differing by the number of H-bonds. These measurements were repeated at different concentrations of trifluoroethanol (TFE)-water mixture, an alpha-helical promoting solvent, in order to check the stability of the conformations determined; no changes were observed up to 50% TFE in solution. Also temperature changes did not produce any variation in the ROESY spectrum in the same condition as above. The NMR and circular dichroism data strongly indicate the presence of an ordered (not alpha-helix nor beta-sheet) highly flexible structure in the eight amino acids repetitive units of SPR3, confirming the prediction of one possible beta-turn per each repeating unit. Thus, biochemical and biophysical data, strongly support SPR3 to function as a flexible cross-bridging protein to provide tensile strength or rigidity to the CE of the stratified squamous epithelia in which it is expressed.

Transglutaminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activation by proteolytic processing.

Lamellar ichthyosis is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. It is caused by mutations in the TGM1 gene that encodes the transglutaminase 1 (TGase 1) enzyme, which is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. TGase 1 is a complex enzyme existing as both cytosolic and membrane-bound forms. Moreover, TGase 1 is proteolytically processed, and the major functionally active form consists of a membrane-bound 67/33/10-kDa complex with a myristoylated and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. To understand better how point mutations, deletions, and truncations found in lamellar ichthyosis disease affect the structure and function of TGase 1, we have expressed in baculovirus and keratinocytes a number of reported TGase 1 mutants. The structural implications of these mutations were examined using a homology-derived three-dimensional model of TGase 1 generated from the known x-ray structure of the related coagulation factor XIIIa enzyme. The present studies demonstrate that loss of TGase 1 activity is not restricted to mutations that directly affect the enzymatic activity. We report a new class of mutations that impair the subsequent post-synthetic processing of the protein into its highly active functional forms.

Carnitine and derivatives in the central nervous system of chick embryo.

1. Carnitine contents and the activity of carnitine acetyltransferase in the egg, in the embryo, and in different brain areas of central nervous system in chick embryo were determined in the course of development. 2. The egg showed low levels of free carnitine and acetylcarnitine. 3. In the whole embryo, at first stages of development, long chain acylcarnitine and acetylcarnitine were the best represented classes of carnitines. 4. In the brain regions acetylcarnitine levels, high at the first days, showed a continual decrease during development. 5. The activity of carnitine acetyltransferase increased and was totally related to development.