Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation

<p>Abstract</p> <p>Background</p> <p>Many studies implicate Arf6 activity in Rac-mediated membrane ruffling and cytoskeletal reorganization. Although Arf6 facilitates the trafficking of Rac1 to the plasma membrane and in many cases Arf6 activation leads to the activation of Rac1, the details of how Arf6 influences Rac function remain to be elucidated.</p> <p>Results</p> <p>We demonstrate in binding assays and by co-immunoprecipitation that GDP-bound Arf6 binds to Kalirin5, a Rho family guanine nucleotide exchange factor, through interaction with the spectrin repeat region. In cells, expression of wild type Arf6 recruits spectrin repeat 5 and Kalirin to the plasma membrane and leads to enhanced Kalirin5-induced ruffling. By contrast, expression of an Arf6 mutant that cannot become activated, Arf6 T27N, still recruits spectrin repeat 5 and Kalirin to membranes but inhibits Kalirin5-induced ruffling in HeLa cells. Kalirin5-induced Rac1 activation is increased by the expression of wild type Arf6 and decreased by Arf6T27N. Furthermore, expression of a catalytically-inactive mutant of Kalirin5 inhibits cytoskeletal changes observed in cells expressing EFA6, an Arf6 guanine nucleotide exchange factor that leads to activation of Rac.</p> <p>Conclusion</p> <p>We show here with over-expressed proteins that the GDP-bound form of Arf6 can bind to the spectrin repeat regions in Kalirin Rho family GEFs thereby recruiting Kalirin to membranes. Although Kalirin is recruited onto membranes by Arf6-GDP, subsequent Rac activation and membrane ruffling requires Arf6 activation. From these results, we suggest that Arf6 can regulate through its GTPase cycle the activation of Rac.</p

TurbEFA: an interdisciplinary effort to investigate the turbulent flow across a forest clearing

the atmosphere within turbulence closure models is mainly limited by a realistic three-dimensional (3D) representation of the vegetation architecture. Within this contribution we present a method to record the 3D vegetation structure and to use this information to derive model parameters that are suitable for numerical flow models. A mixed conifer forest stand around a clearing was scanned and represented by a dense 3D point cloud applying a terrestrial laser scanner. Thus, the plant area density (PAD) with a resolution of one cubic meter was provided for analysis and for numerical simulations. Multi-level high-frequency wind velocity measurements were recorded simultaneously by 27 ultrasonic anemometers on 4 towers for a period of one year. The relationship between wind speed, Reynolds stress and PAD was investigated and a parametrization of the drag coefficient CD by the PAD is suggested. The derived 3D vegetation model and a simpler model (based on classical forest assessments of the site) were applied in a boundary layer model (BLM) and in large-eddy simulations (LES). The spatial development of the turbulent flow over the clearing is further demonstrated by the results of a wind tunnel experiment. The project showed, that the simulation results were improved significantly by the usage of realistic vegetation models. 3D simulations are necessary to depict the influence of heterogeneous canopies on the turbulent flow. Whereas we found limits for the mapping of the vegetation structure within the wind tunnel, there is a considerable potential for numerical simulations. The field measurements and the LES gave new insight into the turbulent flow in the vicinity and across the clearing. The results show that the zones of intensive turbulence development can not be restricted to the locations found in previous studies with more idealized canopies

Directed evolution of an enantioselective Bacillus subtilis lipase

Chiral compounds are of steadily increasing importance to the chemical industry, in particular for the production of pharmaceuticals. Where do these compounds come from? Apart from natural resources, two synthetic strategies are available: asymmetric chemical catalysis using transition metal catalysts and biocatalysis using enzymes. In the latter case, screening programs have identified a number of enzymes. However, their enantioselectivity is often not high enough for a desired reaction. This problem can be solved by applying directed evolution to create enantioselective enzymes as shown here for a lipase from Bacillus subtilis. The reaction studied was the asymmetric hydrolysis of meso-1,4-diacetoxy-2-cyclopentene with the formation of chiral alcohols which were detected by electrospray ionization mass spectrometry. Iterative cycles of random mutagenesis and screening allowed the identification of several variants with improved enantioselectivities. In parallel, we have started to use X-ray structural data to simulate the Bacillus subtilis lipase A-catalyzed substrate hydrolysis by using quantum mechanical and molecular mechanical calculations. This combined approach should finally enable us to devise more efficient strategies for the directed evolution of enantioselective enzymes

TurbEFA: Ein interdisziplinärer Ansatz zur Untersuchung der turbulenten Strömung an einer Waldlichtung

Waldökosysteme spielen eine bedeutende Rolle in der Interaktion zwischen Landoberfläche und Atmosphäre. Ein besseres Verständnis der Austauschprozesse ist unter anderem notwendig für eine Einschätzung der Absorption und Emission von Spurenstoffen (z.B. CO2) und der Risiken von Waldschäden durch Wind, Frost und Dürre. Heutige Studien zur Rolle von terrestrischen Ökosystemen im Wasser- und Kohlenstoffkreislauf basieren auf langfristigen Messungen des Energie- und Massenaustausches zwischen Vegetation und Atmosphäre durch die Eddy-Kovarianz Methode (Goulden et al. 1996). Mehr als 500 Standorte weltweit sind derzeit in FLUXNET organisiert, einem internationalen Netzwerk (Baldocchi et al. 2001) zur kontinuierliche Messungen des Stoff- und Energieaustausches nach standardisierten Methoden (Aubinet et al. 2000). Der Austausch von Waldökosystemen wird dabei an einem Messturm durch eine Punktmessung über dem Bestand bestimmt, die eine bestimmte häufig komplexe Quellfläche repräsentiert. Mehr als drei Dekaden der Forschung in Feldexperimenten und Modellierung haben gezeigt, dass verbleibende Unsicherheiten vor allem durch räumliche Inhomogenität des Austausches begründet sind. Insbesondere fehlen Ansätze für eine geeignete Parametrisierung dieser Inhomogenitäten in numerischen Modellen

HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling

POMC: The Physiological Power of Hormone Processing.

Pro-opiomelanocortin (POMC) is the archetypal polypeptide precursor of hormones and neuropeptides. In this review, we examine the variability in the individual peptides produced in different tissues and the impact of the simultaneous presence of their precursors or fragments. We also discuss the problems inherent in accurately measuring which of the precursors and their derived peptides are present in biological samples. We address how not being able to measure all the combinations of precursors and fragments quantitatively has affected our understanding of the pathophysiology associated with POMC processing. To understand how different ratios of peptides arise, we describe the role of the pro-hormone convertases (PCs) and their tissue specificities and consider the cellular processing pathways which enable regulated secretion of different peptides that play crucial roles in integrating a range of vital physiological functions. In the pituitary, correct processing of POMC peptides is essential to maintain the hypothalamic-pituitary-adrenal axis, and this processing can be disrupted in POMC-expressing tumors. In hypothalamic neurons expressing POMC, abnormalities in processing critically impact on the regulation of appetite, energy homeostasis, and body composition. More work is needed to understand whether expression of the POMC gene in a tissue equates to release of bioactive peptides. We suggest that this comprehensive view of POMC processing, with a focus on gaining a better understanding of the combination of peptides produced and their relative bioactivity, is a necessity for all involved in studying this fascinating physiological regulatory phenomenon

ProSAAS-Derived Peptides are Colocalized with Neuropeptide Y and Function as Neuropeptides in the Regulation of Food Intake

ProSAAS is the precursor of a number of peptides that have been proposed to function as neuropeptides. Because proSAAS mRNA is highly expressed in the arcuate nucleus of the hypothalamus, we examined the cellular localization of several proSAAS-derived peptides in the mouse hypothalamus and found that they generally colocalized with neuropeptide Y (NPY), but not α-melanocyte stimulating hormone. However, unlike proNPY mRNA, which is upregulated by food deprivation in the mediobasal hypothalamus, neither proSAAS mRNA nor proSAAS-derived peptides were significantly altered by 1–2 days of food deprivation in wild-type mice. Furthermore, while proSAAS mRNA levels in the mediobasal hypothalamus were significantly lower in Cpefat/fat mice as compared to wild-type littermates, proNPY mRNA levels in the mediobasal hypothalamus and in other subregions of the hypothalamus were not significantly different between wild-type and Cpefat/fat mice. Intracerebroventricular injections of antibodies to two proSAAS-derived peptides (big LEN and PEN) significantly reduced food intake in fasted mice, while injections of antibodies to two other proSAAS-derived peptides (little LEN and little SAAS) did not. Whole-cell patch clamp recordings of parvocellular neurons in the hypothalamic paraventricular nucleus, a target of arcuate NPY projections, showed that big LEN produced a rapid and reversible inhibition of synaptic glutamate release that was spike independent and abolished by blocking postsynaptic G protein activity, suggesting the involvement of a postsynaptic G protein-coupled receptor and the release of a retrograde synaptic messenger. Taken together with previous studies, these findings support a role for proSAAS-derived peptides such as big LEN as neuropeptides regulating food intake

Efficacy and safety of immune checkpoint inhibitor rechallenge in individuals with hepatocellular carcinoma

BACKGROUND & AIMS: We investigated the efficacy and safety of immune checkpoint inhibitor (ICI) rechallenge in patients with hepatocellular carcinoma (HCC) who received ICI-based therapies in a previous systemic line. METHODS: In this international, retrospective multicenter study, patients with HCC who received at least two lines of ICI-based therapies (ICI-1, ICI-2) at 14 institutions were eligible. The main outcomes included best overall response and treatment-related adverse events. RESULTS: Of 994 ICI-treated patients screened, a total of 58 patients (male, n = 41; 71%) with a mean age of 65.0±9.0 years were included. Median systemic treatment lines of ICI-1 and ICI-2 were 1 (range, 1-4) and 3 (range, 2-9), respectively. ICI-based therapies used at ICI-1 and ICI-2 included ICI alone (ICI-1, n = 26, 45%; ICI-2, n = 4, 7%), dual ICI regimens (n = 1, 2%; n = 12, 21%), or ICI combined with targeted therapies/anti-VEGF (n = 31, 53%; n = 42, 72%). Most patients discontinued ICI-1 due to progression (n = 52, 90%). Objective response rate was 22% at ICI-1 and 26% at ICI-2. Responses at ICI-2 were also seen in patients who had progressive disease as best overall response at ICI-1 (n = 11/21; 52%). Median time-to-progression at ICI-1 and ICI-2 was 5.4 (95% CI 3.0-7.7) months and 5.2 (95% CI 3.3-7.0) months, respectively. Treatment-related adverse events of grade 3-4 at ICI-1 and ICI-2 were observed in 9 (16%) and 10 (17%) patients, respectively. CONCLUSIONS: ICI rechallenge was safe and resulted in a treatment benefit in a meaningful proportion of patients with HCC. These data provide a rationale for investigating ICI-based regimens in patients who progressed on first-line immunotherapy in prospective trials. IMPACT AND IMPLICATIONS: Therapeutic sequencing after first-line immune checkpoint inhibitor (ICI)-based therapy for advanced hepatocellular carcinoma (HCC) remains a challenge as no available second-line treatment options have been studied in immunotherapy-pretreated patients. Particularly, the role of ICI rechallenge in patients with HCC is unclear, as data from prospective trials are lacking. We investigated the efficacy and safety of ICI-based regimens in patients with HCC pretreated with immunotherapy in a retrospective, international, multicenter study. Our data provide the rationale for prospective trials investigating the role of ICI-based regimens in patients who have progressed on first-line immunotherapy

The copper centers of tyramine β-monooxygenase and its catalytic-site methionine variants: an X-ray absorption study

Tyramine β-monooxygenase (TBM) is a member of a family of copper monooxygenases containing two noncoupled copper centers, and includes peptidylglycine monooxygenase and dopamine β-monooxygenase. In its Cu(II) form, TBM is coordinated by two to three His residues and one to two non-His O/N ligands consistent with a [CuM(His)2(OH2)2–CuH(His)3(OH2)] formulation. Reduction to the Cu(I) state causes a change in the X-ray absorption spectroscopy (XAS) spectrum, consistent with a change to a [CuM(His)2S(Met)–CuH(His)3] environment. Lowering the pH to 4.0 results in a large increase in the intensity of the Cu(I)–S extended X-ray absorption fine structure (EXAFS) component, suggesting a tighter Cu–S bond or the coordination of an additional sulfur donor. The XAS spectra of three variants, where the CuM Met471 residue had been mutated to His, Cys, and Asp, were examined. Significant differences from the wild-type enzyme are evident in the spectra of the reduced mutants. Although the side chains of His, Cys, and Asp are expected to substitute for Met at the CuM site, the data showed identical spectra for all three reduced variants, with no evidence for coordination of residue 471. Rather, the K-edge data suggested a modest decrease in coordination number, whereas the EXAFS indicated an average of two His residues at each Cu(I) center. These data highlight the unique role of the Met residue at the CuM center, and pose interesting questions as to why replacement by the cuprophilic thiolate ligand leads to detectable activity whereas replacement by imidazole generates inactive TBM

Neuropeptidomics of the Supraoptic Rat Nucleus

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characteriza-tion of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not im-mediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected