Identification of an insulin-regulated lysophospholipase with homology to neuropathy target esterase

Neuropathy target esterase (NTE) is a member of the family of patatin domain-containing proteins and exhibits phospholipase activity in brain and cultured cells. NTE was originally identified as target enzyme for organophosphorus compounds that cause a delayed paralyzing syndrome with degeneration of nerve axons. Here we show that the structurally related murine protein NTE-related esterase (NRE) is a potent lysophospholipase. The enzyme efficiently hydrolyzes sn-1 esters in lysophosphatidylcholine and lysophosphatidic acid. No lipase activity was observed when triacylglycerols, cholesteryl esters, retinyl esters, phosphatidylcholine, or monoacylglycerol were used as substrates. Although NTE is predominantly expressed in the nervous system, we found the highest NRE mRNA levels in testes, skeletal muscle, cardiac muscle, and adipose tissue. Induction of NRE mRNA concentrations in these tissues during fasting suggested a nutritional regulation of enzyme expression and, in accordance with this observation, insulin reduced NRE mRNA levels in a dose-dependent manner in 3T3-L1 adipocytes. A green fluorescent protein-NRE fusion protein colocalized to the endoplasmic reticulum and lipid droplets. Thus, NRE is a previously unrecognized ER- and lipid droplet-associated lysophospholipase. Regulation of enzyme expression by the nutritional status and insulin suggests a role of NRE in the catabolism of lipid precursors and/or mediators that affect energy metabolism in mammals.     

Altered ion transporter expression in bronchial epithelium in mountaineers with high-altitude pulmonary edema.

Hypoxia inhibits activity and expression of transport proteins of cultured lung alveolar epithelial cells. Here we tested whether hypoxia at high altitude affected the expression of ion transport proteins in tissues obtained from controls and mountaineers with high-altitude pulmonary edema (HAPE) at the Capanna Margherita (4,559 m). Expression was determined by RT-PCR and Western blots from brush biopsies of bronchial epithelium and from leukocytes obtained before and during the stay at high altitude. At low altitude, amounts of mRNAs were not different between control and HAPE-susceptible subjects. At high altitude, the amount of mRNA of Na-K-ATPase, CFTR, and beta-actin of brush biopsies did not change in controls but decreased significantly (-60%) in HAPE-susceptible subjects. There was no change in Na channel mRNAs at high altitude in controls and HAPE. No statistically significant correlation was found between the expression of Na transporters and PO2 and O2 saturation. In leukocytes, 28S-rRNA and Na-K-ATPase decreased at altitude in control and HAPE-susceptible subjects, but no significant change in Na-K-ATPase protein was found. Hypoxia-inducible factor-1alpha mRNA and GAPDH mRNA tended to increase in leukocytes obtained from HAPE-susceptible subjects at high altitude but did not change in controls. These results show that hypoxia induces differences in mRNA expression of ion transport-related proteins between HAPE-susceptible and control subjects but that these changes may not necessarily predict differences in protein concentration or activity. It is therefore unclear whether these differences are related to the pathophysiology of HAPE.     

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔL_k = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membrane-spanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7, 8, 10, 30, 41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site- and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5′ end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3′-OH generated at nic and the subsequent DNA unwinding stage remain obscure.Equally little is known about the process of nucleoprotein uptake by the transport channel. DNA-independent translocation of the relaxases TrwC (R388), MobA (RSF1010), and VirD2 (Ti plasmid) has been demonstrated; thus, current models propose that the relaxase component of the protein-DNA adduct is the substrate actively secreted by the transport system after interaction with the T4CP (42, 66). Cotransport of the covalently linked single-stranded T strand occurs concurrently (42). The mechanisms underlying relaxosome recognition by T4CPs are not understood. Direct interactions have been observed biochemically between the RP4 TraG protein and relaxase proteins of the cognate plasmid (65) and heterologous relaxosomes that it mobilizes (73, 76). TrwB of R388 interacts in vitro with relaxase TrwC and an auxiliary component, TrwA (44). TraD proteins of plasmid R1 and F are known to interact with the auxiliary relaxosome protein TraM (20) via a cluster of C-terminal amino acids (3, 62). Extensive mutagenic analyses (45) plus recent three-dimensional structural data for a complex of the TraM tetramerization domain and the C-terminal tail of TraD (46) have provided more detailed models for the intermolecular contacts involved in recognition.Application of the Cre recombinase assay for translocation of conjugative relaxases as well as effector proteins to eukaryotic cells is currently the most promising approach to elucidate protein motifs recognized by T4CPs (56, 68, 78, 79). Despite that progress, the nature of the interactions between a T4CP and its target protein that initiate secretion and the mechanisms controlling this step remain obscure. In contrast to systems dedicated specifically to effector protein translocation, conjugation systems mobilize nucleoprotein complexes that additionally exhibit catalytic activities, which can be readily monitored. These models are therefore particularly well suited to investigate aspects of regulation occurring at the physical interface of a T4CP and its secretion substrate. For this purpose the MOB_F family of DNA-mobilizing systems is additionally advantageous, since DNA processing within this family features the fusion of a dedicated conjugative helicase to the DNA transesterase enzyme within a single bifunctional protein. The TraI protein of F-like plasmids, originally described as Escherichia coli DNA helicase I (1, 2, 23), and the related TrwC protein of plasmid R388 (25) are well characterized (reviewed in reference 18). Early work by Llosa et al. revealed a complex domain arrangement for TrwC (43). Similar analyses with TraI identified nonoverlapping transesterase and helicase domains (6, 77), while the remaining intermediate and C-terminal regions of the protein additionally provide functions essential to effective conjugative transfer (49, 71). The ability to physically separate the catalytic domains of TraI and TrwC has facilitated a detailed biochemical characterization of their DNA transesterase, ATPase, and DNA-unwinding reactions. Nonetheless, failure of the physically disjointed polypeptides to complement efficient conjugative transfer when coexpressed indicates a role(s) for these proteins in the strand transfer process that goes beyond the need for their dual catalytic activities (43, 50). The assignment of additional functional properties to regions within TraI is a focus of current investigation (16, 29, 49).In all systems studied thus far, conditions used to reconstitute relaxosomes on a supercoiled oriT plasmid have not supported the initiation steps necessary to enable duplex unwinding by a conjugative helicase. The question remains open whether additional protein components are required and/or whether the pathway of initiation is subject to specific repression. In the present study, we applied the IncFII plasmid R1 paradigm to investigate the potential for interaction between purified components of the relaxosome and its cognate T4CP, TraD, to exert regulatory effects on relaxosome activities in vitro. In this and in the accompanying report (72), we present evidence for wide-ranging stimulatory effects of the cytoplasmic domain of TraD protein and its interaction partner TraM on multiple aspects of relaxosome function.     

Acute in vitro hypoxia and high-altitude (4,559 m) exposure decreases leukocyte oxygen consumption

Hypoxia impairs metabolic functions by decreasing activity and expression of ATP-consuming processes. To separate hypoxia from systemic effects, we tested whether hypoxia at high altitude affects basal and PMA-stimulated leukocyte metabolism and how this compares to acute (15 min) and 24 h of in vitro hypoxia. Leukocytes were prepared at low altitude and ～24 h after arrival at 4559 m. Mitochondrial oxygen consumption (JO?) was measured by respirometry, oxygen radicals by electron spin resonance spectroscopy, both at a Po? = 100 mmHg (JO?,???) and 20 mmHg (JO?,??). Acute hypoxia of leukocytes decreased JO? at low altitude. Exposure to high altitude decreased JO?,???, whereas JO?,?? was not affected. Acute hypoxia of low-altitude samples decreased the activity of complexes I, II, and III. At high altitude, activity of complexes I and III were decreased when measured in normoxia. Stimulation of leukocytes with PMA increased JO?,??? at low (twofold) and high altitude (five-fold). At both locations, PMA-stimulated JO? was decreased by acute hypoxia. Basal and PMA-stimulated reactive oxygen species (ROS) production were unchanged at high altitude. Separate in vitro experiments performed at low altitude show that ～75% of PMA-induced increase in JO? was due to increased extra-mitochondrial JO? (JO?(,res); in the presence of rotenone and antimycin A). JO?(,res) was doubled by PMA. Acute hypoxia decreased basal JO?(,res) by ～70% and PMA-stimulated JO?(,res) by about 50% in cells cultured in normoxia and hypoxia (1.5% O?; 24 h). Conversely, 24 h in vitro hypoxia decreased mitochondrial JO?,??? and JO?,??, extra-mitochondrial, basal, and PMA-stimulated JO? were not affected. These results show that 24 h of high altitude but not 24 h in vitro hypoxia decreased basal leukocyte metabolism, whereas PMA-induced JO? and ROS formation were not affected, indicating that prolonged high-altitude hypoxia impairs mitochondrial metabolism but does not impair respiratory burst. In contrast, acute hypoxia impairs respiratory burst at either altitude.     

Clinical Features,Treatment and Outcome of Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma of the Ocular Adnexa: Single Center Experience of 60 Patients

Background

Orbital marginal zone B-cell lymphoma (OAML) constitutes for the most frequent diagnosis in orbital lymphoma. Relatively little data, however, have been reported in larger cohorts of patients staged in a uniform way and no therapy standard exists to date.

Material and Methods

We have retrospectively analyzed 60 patients diagnosed and treated at our institution 1999–2012. Median age at diagnosis was 64 years (IQR 51–75) and follow-up time 43 months (IQR 16–92). All patients had undergone uniform extensive staging and histological diagnosis was made by a reference pathologist according to the WHO classification.

Results

The majority of patients presented with stage IE (n = 40/60, 67%), three had IIE/IIIE and the remaining 17 stage IVE. Seven patients with IVE had bilateral orbital disease whereas the others showed involvement of further organs. Treatment data were available in 58 patients. Local treatment with radiotherapy (14/58, 24%) or surgery (3/58, 5%) resulted in response in 82% of patients. A total of 26 patients (45%) received systemic treatment with a response rate of 85%. Nine patients received antibiotics as initial therapy; response rate was 38%. Watchful-waiting was the initial approach in 6/58 patients. In total 28/58 patients (48%) progressed and were given further therapy. Median time-to-progression in this cohort was 20 months (IQR 9–39). There was no difference in time-to-progression after first-line therapy between the different therapy arms (p = 0.14). Elevated beta-2-microglobulin, plasmacytic differentiation, autoimmune disorder and site of lymphoma were not associated with a higher risk for progress.

Conclusion

Our data underscore the excellent prognosis of OAML irrespective of initial therapy, as there was no significant difference in time-to-progression and response between local or systemic therapy. In the absence of randomized trials, the least toxic individual approach should be chosen for OAML.     

Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.     

Monoacylglycerol Lipases Act as Evolutionarily Conserved Regulators of Non-oxidative Ethanol Metabolism

Fatty acid ethyl esters (FAEEs) are non-oxidative metabolites of ethanol that accumulate in human tissues upon ethanol intake. Although FAEEs are considered as toxic metabolites causing cellular dysfunction and tissue damage, the enzymology of FAEE metabolism remains poorly understood. In this study, we used a biochemical screen in Saccharomyces cerevisiae to identify and characterize putative hydrolases involved in FAEE catabolism. We found that Yju3p, the functional orthologue of mammalian monoacylglycerol lipase (MGL), contributes >90% of cellular FAEE hydrolase activity, and its loss leads to the accumulation of FAEE. Heterologous expression of mammalian MGL in yju3Δ mutants restored cellular FAEE hydrolase activity and FAEE catabolism. Moreover, overexpression or pharmacological inhibition of MGL in mouse AML-12 hepatocytes decreased or increased FAEE levels, respectively. FAEEs were transiently incorporated into lipid droplets (LDs) and both Yju3p and MGL co-localized with these organelles. We conclude that the storage of FAEE in inert LDs and their mobilization by LD-resident FAEE hydrolases facilitate a controlled metabolism of these potentially toxic lipid metabolites.     

Insulin receptor kinase‐independent signaling via tyrosine phosphorylation of phosphatase PHLPP1

Most insulin responses correlate well with insulin receptor (IR) Tyr kinase activation; however, critical exceptions to this concept have been presented. Specific IR mutants and stimulatory IR antibodies demonstrate a lack of correlation between IR kinase activity and specific insulin responses in numerous independent studies. IR conformation changes in response to insulin observed with various IR antibodies define an IR kinase‐independent signal that alters the C‐terminus. IR‐related receptors in lower eukaryotes that lack a Tyr kinase point to an alternative mechanism of IR signaling earlier in evolution. However, the implied IR kinase‐independent signaling mechanism remained obscure at the molecular level. Here we begin to define the molecular basis of an IR‐dependent but IR kinase‐independent insulin signal that is equally transmitted by a kinase‐inactive mutant IR. This insulin signal results in Tyr phosphorylation and catalytic activation of phosphatase PHLPP1 via a PI 3‐kinase‐independent, wortmannin‐insensitive signaling pathway. Dimerized SH2B1/PSM is a critical activator of the IR kinase and the resulting established insulin signal. In contrast it is an inhibitor of the IR kinase‐independent insulin signal and disruption of SH2B1/PSM dimer binding to IR potentiates this signal. Dephosphorylation of Akt2 by PHLPP1 provides an alternative, SH2B1/PSM‐regulated insulin‐signaling pathway from IR to Akt2 of opposite polarity and distinct from the established PI 3‐kinase‐dependent signaling pathway via IRS proteins. In combination, both pathways should allow the opposing regulation of Akt2 activity at two phosphorylation sites to specifically define the insulin signal in the background of interfering Akt‐regulating signals, such as those controlling cell proliferation and survival. J. Cell. Biochem. 107: 65–75, 2009. © 2009 Wiley‐Liss, Inc.