Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Ubiquitin Carboxy-Terminal Hydrolase L1 (UCH-L1) is increased in cerebrospinal fluid and plasma of patients after epileptic seizure

ABSTRACT: BACKGROUND: Clinical and experimental studies have demonstrated that seizures can cause molecular and cellular responses resulting in neuronal damage. At present, there are no valid tests for assessing organic damage to the brain associated with seizure. The aim of this study was to investigate cerebrospinal fluid (CSF) and plasma concentrations of Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), a sensitive indicator of acute injury to brain neurons, in patients with tonic--clonic or partial secondarily generalized seizures due to various etiologies. METHODS: CSF and plasma concentrations of UCH-L1 were assessed in 52 patients within 48 hours after epileptic seizure and in 19 controls using ELISA assays. RESULTS: CSF obtained within 48 hours after seizure or status epilepticus (SE) presented significantly higher levels of UCH-L1 compared to controls (p = 0.008). Plasma UCH-L1 concentrations were negatively correlated with time to sample withdrawal. An analysis conducted using only the first 12 hours post-seizure revealed significant differences between concentrations of UCH-L1 in plasma and controls (p = 0.025). CSF and plasma concentrations were strongly correlated with age in patients with seizure, but not in control patients. Plasma UCH-L1 levels were also significantly higher in patients after recurrent seizures (n = 4) than in those after one or two seizures (p = 0.013 and p = 0.024, respectively). CONCLUSION: Our results suggest that determining levels of neuronal proteins may provide valuable information on the assessment of brain damage following seizure. These data might allow clinicians to make more accurate therapeutic decisions, to identify patients at risk of progression and, ultimately, to provide new opportunities for monitoring therapy and targeted therapeutic interventions.     

Manipulating the proximal triad His-Asn-Arg in human myeloperoxidase

In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionate substituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (−50 mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidation states showed significant differences. Additionally, rates of ligand binding and oxidation of both one- and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His–Asn–Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate binding site(s) at the entrance to the heme cavity is discussed.     

Nitrogen availability affects saprotrophic basidiomycetes decomposing pine needles in a long term laboratory study

Fungi, especially basidiomycetous litter decomposers, are pivotal to the turnover of soil organic matter in forest soils. Many litter decomposing fungi have a well-developed capacity to translocate resources in their mycelia, a feature that may significantly affect carbon (C) and nitrogen (N) dynamics in decomposing litter. In an eight-month long laboratory study we investigated how the external availability of N affected the decomposition of Scots pine needles, fungal biomass production, N retention and N-mineralization by two litter decomposing fungi – Marasmius androsaceus and Mycena epipterygia. Glycine additions had a general, positive effect on fungal biomass production and increased accumulated needle mass loss after 8 months, suggesting that low N availability may limit fungal growth and activity in decomposing pine litter. Changes in the needle N pool reflected the dynamics of the fungal mycelium. During late decomposition stages, redistribution of mycelium and N out from the decomposed needles was observed for M. epipterygia, suggesting autophagous self degradation.     

Predation by juvenile Platichthys flesus (L.) on shelled prey species in a bare sand and a drift algae habitat

Due to increasing eutrophication of the coastal Baltic waters, drifting algae are a common phenomenon. Drifting algal mats accumulate on shallow sandy bottoms in late summer and autumn, and affect the ambient fauna. Juvenile flounder, Platichthys flesus, utilize these habitats during their first few years. They feed on benthic meio- and macrofauna; part of their diet consists of shelled species, such as Ostracods, and juvenile Hydrobia spp. and Macoma balthica. Earlier studies have shown that up to 75% of ostracods and 92% of hydrobiids survive the gut passage of juvenile flounder, while all M. balthica are digested by the fish. We conducted laboratory experiments to study how the shelled prey responded to a drift algal mat, and the predation efficiency of juvenile P. flesus on these prey species on bare sand and with drifting algae (50% coverage). Hydrobia spp. utilized the drift algae as a habitat and, after 1 h, 50% had moved into the algae; ostracods and M. balthica were more stationary and, after 96 h, only 23 and 12%, respectively, were found in the algae. For the predation efficiency of P. flesus, a two-way ANOVA with habitat (algae, bare sand) and predation (fish, no fish) as factors revealed that both algae and predation affected negatively the survival of all three prey species. The algae, thus, affected the predation efficiency of juvenile P. flesus and the consumption of prey was much reduced in the algal treatments compared to the bare sand. This was due probably to increased habitat complexity and the ability of prey, especially hydrobiids, to use the algal mat as a refuge. Altered habitat structure due to drift algae, together with the resultant changes in habitat (refuge) value for different prey species, may profoundly change the structure of benthic communities.     

Proteome and cytoskeleton responses in osteosarcoma cells with reduced OXPHOS activity

We have recently shown disorganization of the vimentin network in cultured cells deficient in oxidative phosphorylation (OXPHOS). We describe here the cellular responses to OXPHOS deficiency in osteosarcoma cells upon complex I (CI) and complex IV (CIV) inhibition, and upon the lack of mitochondrial DNA (rho0 cells). We examined the cytoskeletal organization and the distribution of mitochondria and analysed total proteome by 2-DE and vimentin expression by ELISA. Upon CIV inhibition and in rho0 cells, the vimentin network had collapsed around the nucleus and formed thick bundles. The mitochondria formed a perinuclear crescent upon CIV inhibition, whereas they accumulated around the nucleus in the rho0 cells, where the amount of vimentin was increased. Analysis of the total proteome revealed that a lack of mitochondrial DNA or inhibition of CI or CIV led to changes in the expression of cytoskeletal and cytoskeleton-associated proteins and proteins involved in apoptosis, OXPHOS, glycolysis, the tricarboxylic acid cycle, and oxidative stress responses. Our findings suggest that a deficiency in the energy converting system and oxidative stress can lead to cytoskeletal changes.     

Surrogate antigens as targets for proteome-wide binder selection

In the past decade, many initiatives were taken for the development of antibodies for proteome-wide studies, as well as characterisation and validation of clinically relevant disease biomarkers. Phage display offers many advantages compared to antibody generation by immunisation because it is an unlimited resource of affinity reagents without batch-to-batch variation and is also amendable for high throughput in contrast to conventional hybridoma technology. One of the major bottlenecks to proteome-wide binder selection is the limited supply of suitable target antigens representative of the human proteome. Here, we provide proof of principle of using easily accessible, cancer-associated protein epitope signature tags (PrESTs), routinely generated within the Human Protein Atlas project, as surrogate antigens for full-length proteins in phage selections for the retrieval of target-specific binders. These binders were subsequently tested in western blot, immunohistochemistry and protein microarray application to demonstrate their functionality.     

Lizard scales in an adaptive radiation: variation in scale number follows climatic and structural habitat diversity in Anolis lizards

Lizard scales vary in size, shape and texture among and within species. The overall function of scales in squamates is attributed to protection against abrasion, solar radiation and water loss. We quantified scale number of Anolis lizards across a large sample of species (142 species) and examined whether this variation was related either to structural or to climatic habitat diversity. We found that species in dry environments have fewer, larger scales than species in humid environments. This is consistent with the hypothesis that scales reduce evaporative water loss through the skin. In addition, scale number varied among groups of ecomorphs and was correlated with aspects of the structural microhabitat (i.e. perch height and perch diameter). This was unexpected because ecomorph groups are based on morphological features related to locomotion in different structural microhabitats. Body scales are not likely to play an important role in locomotion in Anolis lizards. The observed variation may relate to other features of the ecomorph niche and more work is needed to understand the putative adaptive basis of these patterns. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 570–579.