Characterization of constitutive ER-phagy of excess membrane proteins

Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Whereas endogenously expressed ER resident proteins serve as cargos at a basal level, this level can be induced by overexpression of membrane proteins that are not ER residents. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation-induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.     

Diminished Memory T-Cell Expansion Due to Delayed Kinetics of Antigen Expression by Lentivectors

Memory CD8⁺ T lymphocytes play a central role in protective immunity. In attempt to increase the frequencies of memory CD8⁺ T cells, repeated immunizations with viral vectors are regularly explored. Lentivectors have emerged as a powerful vaccine modality with relatively low pre-existing and anti-vector immunity, thus, thought to be ideal for boosting memory T cells. Nevertheless, we found that lentivectors elicited diminished secondary T-cell responses that did not exceed those obtained by priming. This was not due to the presence of anti-vector immunity, as limited secondary responses were also observed following heterologous prime-boost immunizations. By dissecting the mechanisms involved in this process, we demonstrate that lentivectors trigger exceptionally slow kinetics of antigen expression, while optimal activation of lentivector-induced T cells relays on durable expression of the antigen. These qualities hamper secondary responses, since lentivector-encoded antigen is rapidly cleared by primary cytotoxic T cells that limit its presentation by dendritic cells. Indeed, blocking antigen clearance by cytotoxic T cells via FTY720 treatment, fully restored antigen presentation. Taken together, while low antigen expression is expected during secondary immunization with any vaccine vector, our results reveal that the intrinsic delayed expression kinetics of lentiviral-encoded antigen, further dampens secondary CD8⁺ T-cell expansion.     

A rat model of exercise-induced asthma: a nonspecific response to a specific immunogen

Exercise-induced bronchoconstriction (EIB) is common; however, key aspects of its pathogenesis are still unclear. We investigated the feasibility of adapting an established animal model of asthma to investigate the earliest stages of EIB. The hypothesis was that a single exposure to a normally innocuous, and brief, exercise challenge could trigger EIB symptoms in rats previously sensitized to ovalbumin (OVA) but otherwise unchallenged. Brown-Norway rats were sensitized by intraperitoneal injection of OVA at 0 and 2 wk. At week 3, animals were exposed to either aerosolized OVA (SS) or exercise (EXS). A trained, blinded, clinical observer graded EIB by respiratory sounds. Plasma and lung cytokine levels were analyzed. No control rats with or without exercise (EX, CON) showed evidence of EIB. Eighty percent of the SS group demonstrated abnormal breath sounds upon exposure to aerosolized OVA. Approximately 30% of EXS rats sensitized to OVA but exposed only to exercise had abnormal breath sounds. Lung tissue levels of TNF-α, IL-1α, growth-related oncogene/keratinocyte/chemoattractant, and IFN-γ were significantly higher (P < 0.001) in the SS group, relative to all other groups. Changes in most of these cytokines were not notable in the EXS rats, suggesting a different mechanism of EIB. Remarkably, IFN-γ, but not the other cytokines measured, was significantly elevated following brief exercise in both sensitized and unsensitized rats. Exercise led to detectable breathing sound abnormalities in sensitized rats, but less severe than those observed following classical OVA challenge. Precisely how this immune crossover occurs is not known, but this model may be useful in elucidating essential mechanisms of EIB.     

Morphological Heterogeneity and Attachment of Phaeobacter inhibens

The Roseobacter clade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters.     

How reliable are experimental protein-protein interaction data?

Data of protein-protein interactions provide valuable insight into the molecular networks underlying a living cell. However, their accuracy is often questioned, calling for a rigorous assessment of their reliability. The computation offered here provides an intelligible mean to assess directly the rate of true positives in a data set of experimentally determined interacting protein pairs. We show that the reliability of high-throughput yeast two-hybrid assays is about 50%, and that the size of the yeast interactome is estimated to be 10,000-16,600 interactions.     

Altering Neurospora crassa MOB2A exposes its functions in development and affects its interaction with the NDR kinase COT1

The Neurospora crassa Mps One Binder (MOB) proteins MOB2A and MOB2B physically interact with the Nuclear Dbf2 Related (NDR) kinase COT1 and have been shown to have overlapping functions in various aspects of asexual development. Here, we identified two N. crassa MOB2A residues, Tyr117 and Tyr119, which are potentially phosphorylated. Using phosphomimetic mob‐2a mutants we have been able to establish that apart from their previously described roles, MOB2A/B are involved in additional developmental processes. Enhanced conidial germination, accompanied by conidial agglutination, in the phosphomimetic mutants indicated that MOB2A is a negative regulator of germination. Thick‐section imaging of perithecia revealed slow maturation and a lack of asci alignment in the mutant strains demonstrating a role for MOB2A in sexual development. We demonstrate that even though MOB2A and MOB2B have some overlapping functions, MOB2B cannot compensate for the roles MOB2A has in conidiation and germination. Altering Tyr residues 117 and 119 impaired the physical interactions between MOB2A and COT1, most likely contributing to some of the observed effects. As cot‐1 and the phosphomimetic mutants share an extragenic suppressor (gul‐1), we concluded that at least some of the effects imposed by altering Tyr117 and Tyr119 are mediated by the NDR kinase.     

Two faces of chondroitin sulfate proteoglycan in spinal cord repair: a role in microglia/macrophage activation

Background

Chondroitin sulfate proteoglycan (CSPG) is a major component of the glial scar. It is considered to be a major obstacle for central nervous system (CNS) recovery after injury, especially in light of its well-known activity in limiting axonal growth. Therefore, its degradation has become a key therapeutic goal in the field of CNS regeneration. Yet, the abundant de novo synthesis of CSPG in response to CNS injury is puzzling. This apparent dichotomy led us to hypothesize that CSPG plays a beneficial role in the repair process, which might have been previously overlooked because of nonoptimal regulation of its levels. This hypothesis is tested in the present study.

Methods and Findings

We inflicted spinal cord injury in adult mice and examined the effects of CSPG on the recovery process. We used xyloside to inhibit CSPG formation at different time points after the injury and analyzed the phenotype acquired by the microglia/macrophages in the lesion site. To distinguish between the resident microglia and infiltrating monocytes, we used chimeric mice whose bone marrow-derived myeloid cells expressed GFP. We found that CSPG plays a key role during the acute recovery stage after spinal cord injury in mice. Inhibition of CSPG synthesis immediately after injury impaired functional motor recovery and increased tissue loss. Using the chimeric mice we found that the immediate inhibition of CSPG production caused a dramatic effect on the spatial organization of the infiltrating myeloid cells around the lesion site, decreased insulin-like growth factor 1 (IGF-1) production by microglia/macrophages, and increased tumor necrosis factor alpha (TNF-α) levels. In contrast, delayed inhibition, allowing CSPG synthesis during the first 2 d following injury, with subsequent inhibition, improved recovery. Using in vitro studies, we showed that CSPG directly activated microglia/macrophages via the CD44 receptor and modulated neurotrophic factor secretion by these cells.

Conclusions

Our results show that CSPG plays a pivotal role in the repair of injured spinal cord and in the recovery of motor function during the acute phase after the injury; CSPG spatially and temporally controls activity of infiltrating blood-borne monocytes and resident microglia. The distinction made in this study between the beneficial role of CSPG during the acute stage and its deleterious effect at later stages emphasizes the need to retain the endogenous potential of this molecule in repair by controlling its levels at different stages of post-injury repair.     

Prostate cancer PET bioprobes: synthesis of [18F]-radiolabeled hydroxyflutamide derivatives

Approximately 80-90% of prostate cancers are androgen dependent at initial diagnosis. The androgen receptor (AR) is present in most advanced prostate cancer specimens and is believed to have a critical role in its development. Today, treatment of prostate cancer is done by inhibition of AR using antiandrogens such as flutamide (pro-drug of hydroxyflutamide), nilutamide, and bicalutamide. However, there is currently no noninvasive imaging modalities to detect, guide, and monitor specific treatment of AR-positive prostate cancer. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-propanamide [18F]-1 and N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide [18F]-2, derivatives of hydroxyflutamide, were synthesized as a fluorine-containing imaging agent candidates. A three-step fluorine-18 radiosynthesis route was developed, and the compounds were successfully labeled with a 10+/-3% decay corrected radiochemical yield, 95% radiochemical purity, and a specific activity of 1500+/-200 Ci/mmol end of bombardment (n = 10). These labeled biprobes not only may enable for the future quantitative molecular imaging of AR-positive prostate cancer using positron emission tomography but may also allow for image-guided treatment of prostate cancer.