The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein–protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.     

Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets

GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.     

The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.     

An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters

An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40 degrees C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level.     

A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties

Background

Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpG_C3) as module for half-life extension. SpG_C3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.

Methodology/Principal Findings

Using site-directed mutagenesis, we generated a Fab-selective mutant (SpG_C3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpG_C3FabRR) conferred prolonged plasma half-lives compared with SpG_C3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpG_C3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.

Conclusions/Significance

The half-life extension properties of SpG_C3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpG_C3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.     

The cancer secretome,current status and opportunities in the lung,breast and colorectal cancer context

Despite major improvements on the knowledge and clinical management, cancer is still a deadly disease. Novel biomarkers for better cancer detection, diagnosis and treatment prediction are urgently needed. Proteins secreted, shed or leaking from the cancer cell, collectively termed the cancer secretome, are promising biomarkers since they might be detectable in blood or other biofluids. Furthermore, the cancer secretome in part represents the tumor microenvironment that plays a key role in tumor promoting processes such as angiogenesis and invasion. The cancer secretome, sampled as conditioned medium from cell lines, tumor/tissue interstitial fluid or tumor proximal body fluids, can be studied comprehensively by nanoLC-MS/MS-based approaches. Here, we outline the importance of current cancer secretome research and describe the mass spectrometry-based analysis of the secretome. Further, we provide an overview of cancer secretome research with a focus on the three most common cancer types: lung, breast and colorectal cancer. We conclude that the cancer secretome research field is a young, but rapidly evolving research field. Up to now, the focus has mainly been on the discovery of novel promising secreted cancer biomarker proteins. An interesting finding that merits attention is that in cancer unconventional secretion, e.g. via vesicles, seems increased. Refinement of current approaches and methods and progress in clinical validation of the current findings are vital in order to move towards applications in cancer management. This article is part of a Special Issue entitled: An Updated Secretome.     

The Ripoptosome, a signaling platform that assembles in response to genotoxic stress and loss of IAPs

A better understanding of the mechanisms through which anticancer drugs exert their effects is essential to improve combination therapies. While studying how genotoxic stress kills cancer cells, we discovered a large ～2MDa cell death-inducing platform, referred to as "Ripoptosome." It contains the core components RIP1, FADD, and caspase-8, and assembles in response to genotoxic stress-induced depletion of XIAP, cIAP1 and cIAP2. Importantly, it forms independently of TNF, CD95L/FASL, TRAIL, death-receptors, and mitochondrial pathways. It also forms upon Smac-mimetic (SM) treatment without involvement of autocrine TNF. Ripoptosome assembly requires RIP1's kinase activity and can stimulate caspase-8-mediated apoptosis as well as caspase-independent necrosis. It is negatively regulated by FLIP, cIAP1, cIAP2, and XIAP. Mechanistically, IAPs target components of this complex for ubiquitylation and inactivation. Moreover, we find that etoposide-stimulated Ripoptosome formation converts proinflammatory cytokines into prodeath signals. Together, our observations shed new light on fundamental mechanisms by which chemotherapeutics may kill cancer cells.     

Comparison of diagnostic tests for the detection of <Emphasis Type="Italic">Brucella</Emphasis> spp. in camel sera

Background

Brucellosis in livestock causes enormous losses for economies of developing countries and poses a severe health risk to consumers of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. To date, little information on application of real-time PCR for detection of Brucella in camel serum is available. Therefore, this study was performed to compare the diagnostic efficiency of different serological tests and real-time PCR in order to identify the most sensitive, rapid and simple combination of tests for detecting Brucella infection in camels.

Findings

A total of 895 serum samples collected from apparently healthy Sudanese camels was investigated. Sudan is a well documented endemic region for brucellosis with cases in humans, ruminants, and camels. Rose Bengal Test (RBT), Complement Fixation Test (CFT), Slow Agglutination Test (SAT), Competitive Enzyme Linked Immunosorbant Assay (cELISA) and Fluorescence Polarization Assay (FPA) as well as real-time PCR were used. Our findings revealed that bcsp31 kDa real-time PCR detected Brucella DNA in 84.8% (759/895) of the examined samples, of which 15.5% (118/759) were serologically negative. Our results show no relevant difference in sensitivity between the different serological tests. FPA detected the highest number of positive cases (79.3%) followed by CFT (71.4%), RBT (70.7%), SAT (70.6%) and cELISA (68.8%). A combination of real-time PCR with one of the used serological tests identified brucellosis in more than 99% of the infected animals. 59.7% of the examined samples were positive in all serological tests and real-time PCR. A subpopulation of 6.8% of animals was positive in all serological tests but negative in real-time PCR assays. The high percentage of positive cases in this study does not necessarily reflect the seroprevalence of the disease in the country but might be caused by the fact that the camels were imported from brucellosis infected herds of Sudan, accidentally. Seroprevalence of brucellosis in camels should be examined in confirmatory studies to evaluate the importance of brucellosis in this animal species.

Conclusion

We suggest combining bcsp31 real-time PCR with either FPA, CFT, RBT or SAT to screen camels for brucellosis.

     

Binding of the small-molecule kinase inhibitor ruxolitinib to membranes does not disturb membrane integrity

Ruxolitinib is a small-molecule protein kinase inhibitor, which is used as a therapeutic agent against several diseases. Due to its anti-inflammatory impact, ruxolitinib has also been considered recently for usage in the treatment of Covid-19. While the specific effects of ruxolitinib on Janus kinases (JAK) is comparatively well investigated, its (unspecific) impact on membranes has not been studied in detail so far. Therefore, we characterized the interaction of this drug with lipid membranes employing different biophysical approaches. Ruxolitinib incorporates into the glycerol region of lipid membranes causing an increase in disorder of the lipid chains. This binding, however, has only marginal influence on the structure and integrity of membranes as found by leakage and permeation assays.