Necroptosis-like neuronal cell death caused by cellular cholesterol accumulation [Neurobiology]

October 18th, 2016 by Funakoshi, T., Aki, T., Tajiri, M., Unuma, K., Uemura, K.

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mM l-Nor for ~6 hours. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence cyclodextrin (CD) also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells.

The phosphoinositide 3-kinase regulates retrograde trafficking of the iron permease CgFtr1 and iron homeostasis in Candida glabrata [Microbiology]

October 11th, 2016 by Sharma, V., Purushotham, R., Kaur, R.

The phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces PI3P, has been implicated in protein trafficking, intracellular survival and virulence in the pathogenic yeast Candida glabrata. Here, we demonstrate PI3-kinase (CgVps34) to be essential for maintenance of cellular iron homeostasis. We examine how CgVps34 regulates the fundamental process of iron acquisition, and underscore its function in vesicular trafficking as a central determinant. RNA-sequencing analysis revealed iron homeostasis genes to be differentially expressed upon CgVps34 disruption. Consistently, the Cgvps34Δ mutant displayed growth attenuation in low- and high-iron media, increased intracellular iron content, elevated mitochondrial aconitase activity, impaired biofilm formation and extenuated mouse organ colonization potential. Further, we demonstrate for the first time that C. glabrata cells respond to iron-limitation by expressing the iron permease CgFtr1 primarily on the cell membrane, and to iron-excess via internalization of the plasma membrane-localized CgFtr1 to the vacuole. Our data show that CgVps34 is essential for the latter process. We also report that macrophage-internalized C. glabrata cells express CgFtr1 on the cell membrane indicative of an iron-restricted macrophage internal milieu, and Cgvps34Δ cells display better survival in iron-enriched medium-cultured macrophages. Overall, our data reveal the centrality of PI3K signaling in iron metabolism and host colonization.
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Cdc24 is essential for long-range end resection in the repair of dsDNA breaks [Metabolism]

October 11th, 2016 by Zhang, H., Hua, Y., Li, R., Kong, D.

Double-stranded DNA breaks (DSBs) are highly detrimental DNA lesions, which may be repaired by the homologous recombination-mediated repair pathway. The 5 prime to 3 prime direction of long-range end resection on one DNA strand, in which 3 prime-single-strand DNA overhangs are created from broken DNA ends, is an essential step in this pathway. Dna2 has been demonstrated as an essential nuclease in this event, but the molecular mechanism how Dna2 is recruited to DNA break sites in vivo is not elucidated. In this study, a novel recombination factor called Cdc24 was identified in fission yeast. We demonstrated that Cdc24 localizes to DNA break sites during the repair of DNA breaks and is an essential factor for long-range end resection. We also determined that Cdc24 plays a direct role in recruiting Dna2 to DNA break sites through its interaction with Dna2 and replication protein A (RPA). Further, this study revealed that RPA acts as a foundation in assembling the machinery for long-range end resection by its essential role in recruiting Cdc24 and Dna2 to DNA break sites. These results define Cdc24 as an essential factor for long-range end resection in the repair of DSBs, opening the door for further investigations into the enzymes involved in long-range end resection for DSB repair.

Bipartite Role of Hsp90 Keeps CRAF Kinase Poised for Activation [Protein Structure and Folding]

October 5th, 2016 by Mitra, S., Ghosh, B., Gayen, N., Roy, J., Mandal, A. K.

CRAF kinase maintains cell viability, growth and proliferation by participating in MAPK pathway. Unlike BRAF, CRAF requires continuous chaperoning by Hsp90 to retain MAPK signaling. But, the reason behind the continuous association of Hsp90 with CRAF is still elusive. In this study, we have identified the bipartite role of Hsp90 in chaperoning CRAF kinase. Hsp90 facilitates Ser-621 phosphorylation of CRAF and prevents the kinase from degradation. Co-chaperone Cdc37 assists in this phosphorylation event. However, after folding the stability of the kinase becomes insensitive to Hsp90 inhibition, although the physical association between Hsp90 and CRAF remains intact. We observe that over-expression of Hsp90 stimulates MAPK signaling by activating CRAF. The interaction between Hsp90 and CRAF is substantially increased under elevated level of cellular Hsp90 and in presence of either active Ras (RasV12) or EGF. Surprisingly, enhanced binding of Hsp90 to CRAF occurs prior to the Ras-CRAF association and facilitates actin recruitment to CRAF for efficient Ras-CRAF interaction, which is independent of Hsp90s ATPase activity. However, monomeric CRAF (CRAF R401H) shows abrogated interaction with both Hsp90 and actin, thereby affecting Hsp90-dependent CRAF activation. This finding suggests that stringent assemblage of Hsp90 keeps CRAF kinase equipped for participating in MAPK pathway. Thus, the role of Hsp90 in CRAF maturation and activation acts as a limiting factor to maintain the function of a strong client like CRAF kinase.

N6-methyladenosine seqencing highlights the involvement of mRNA methylation in oocyte meiotic maturation and embryo development by regulating translation in Xenopus laevis [Developmental Biology]

September 9th, 2016 by Qi, S.-T., Ma, J.-Y., Wang, Z.-B., Guo, L., Hou, Y., Sun, Q.-Y.

During the oogenesis of Xenopus laevis, oocytes accumulate maternal materials for early embryo development. As the transcription activity of oocyte is silenced at the fully-grown stage and the global genome is reactivated only by the mid-blastula embryo stage, the translation of maternal mRNAs accumulated during oocyte growth should be accurately regulated. Previous evidence has illustrated that the poly(A) tail length and RNA binding elements mediate RNA translation regulation in oocyte. Recently, RNA methylation is found to exist in various systems. In the present study, we sequenced the N6-methyladenosine (m6A) modified mRNAs in fully-grown germinal vesicle (GV) stage and metaphase II (MII) stage oocytes. As a result, we identified 4207 mRNAs with m6A peaks in the GV stage or MII stage oocytes. When we integrated the mRNA methylation data with transcriptome and proteome data, we found that the highly methylated mRNAs showed significantly lower protein levels than those of the hypomethylated mRNAs, although the RNA levels showed no significant difference. We also found that the hypomethylated mRNAs were mainly enriched in the cell cycle and translation pathways, whereas the highly methylated mRNAs were mainly associated with the protein phosphorylation. Our results suggest that the oocyte mRNA methylation can regulate the cellular translation and cell division during oocyte meiotic maturation and early embryo development.
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Identification of the plant ribokinase and discovery of a role for Arabidopsis ribokinase in nucleoside metabolism [Plant Biology]

September 6th, 2016 by Riggs, J. W., Rockwell, N. C., Cavales, P. C., Callis, J.

Ribose can be used for energy or as a component of several important biomolecules but in order for it to be used in either capacity it must first be phosphorylated by ribokinase (RBSK). RBSK proteins are part of the phosphofructokinase-B (pfkB) family of carbohydrate kinases. Sequence comparisons of pfkB proteins from the model plant Arabidopsis thaliana with the human and E. coli RBSK identified a single candidate RBSK, At1g17160 (AtRBSK). AtRBSK is more similar to predicted RBSKs from other plant species and to known mammalian and prokaryotic RBSK than to all other PfkB proteins in Arabidopsis. AtRBSK contains a predicted chloroplast transit peptide, and we confirmed plastid localization using AtRBSK fused to YFP. Structure prediction software verified that the AtRBSK sequence mapped onto a known RBSK structure. Kinetic parameters of purified recombinant AtRBSK were determined to be Kmribose = 153 μM +/- 17 μM, KmATP = 45.9 μM +/- 5.6 μM, kcat = 2.0 s-1. Substrate inhibition was observed for AtRBSK (KiATP = 2.44 mM +/- 0.36 mM), as has been demonstrated for other RBSK proteins. Ribose accumulated in Arabidopsis plants lacking AtRBSK. Such plants grew normally unless media was supplemented with ribose, which led to chlorosis and growth inhibition. Ribose accumulated in plants lacking AtRBSK. Both chlorosis and ribose accumulation were abolished upon the introduction of a transgene expressing AtRBSK-MYC, demonstrating that the loss of protein is responsible for the ribose hypersensitivity. Ribose accumulation in plants lacking AtRBSK was reduced in plants also deficient in the nucleoside ribohydrolase NSH1, linking AtRBSK activity to nucleoside metabolism.

Structures of a Nonribosomal Peptide Synthetase Module Bound to MbtH-Like Proteins Support a Highly Dynamic Domain Architecture [Protein Structure and Folding]

September 5th, 2016 by Miller, B. R., Drake, E. J., Shi, C., Aldrich, C. C., Gulick, A. M.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of peptide natural products. During synthesis, the multidomain NRPSs act as an assembly line, passing the growing product from one module to the next. Each module generally consists of an integrated peptidyl carrier protein (PCP), an amino acid-loading adenylation domain, and a condensation domain that catalyzes peptide bond formation. Some adenylation domains interact with small partner proteins called MbtH-like proteins (MLPs) that enhance solubility or activity. A structure of an MLP bound to an adenylation domain has been previously reported using a truncated adenylation domain, precluding any insight that might derive from understanding the influence of the MLP on the intact adenylation domain or on the dynamics of the entire NRPS module. Here, we present the structures of the full length NRPS EntF bound to the MLPs from E. coli and Pseudomonas aeruginosa. These new structures, along with biochemical and bioinformatic support, further elaborate the residues that define the MLP-adenylation domain interface. Additionally the structures highlight the dynamic behavior of NRPS modules, including the module core formed by the adenylation and condensation domains as well as the orientation of the mobile thioesterase domain.
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Leptin Enhances TH2 and ILC2 Responses in Allergic Airway Disease [Molecular Bases of Disease]

August 26th, 2016 by Zheng, H., Zhang, X., Castillo, E. F., Luo, Y., Liu, M., Yang, X. O.

Allergic asthma and obesity are the leading health problems in the world. Many studies have shown that obesity is a risk factor of development of asthma. However, the underlying mechanism has not been well established. In this study, we demonstrate that leptin, an adipokine elevated in obese individuals, promoted proliferation and survival of pro-allergic type 2 helper T cells and group 2 innate lymphoid cells and production of type 2 cytokines, which together contribute to allergic responses. Leptin activates mTORC1, MAPK and STAT3 pathways in TH2 cells. The effects of leptin on TH2 cell proliferation, survival and cytokine production are dependent on the mTORC1 and MAPK pathways as revealed by specific inhibitors. In vivo, leptin-deficiency led to attenuated experimental allergic airway inflammation. Our results thus support that obesity associated elevation of leptin may increase the susceptibility of asthma via modulation of pro-allergic lymphocyte responses.

Proof of Principle: Coronin 1A – An Intrinsic Modulator of T Lymphocyte Function [Signal Transduction]

August 26th, 2016 by Siegmund, K., Klepsch, V., Hermann-Kleiter, N., Baier, G.

Coronins are evolutionarily conserved proteins that were originally identified as modulators of actin-dependent processes. Studies analyzing complete Coronin 1a knockout mice have shown that this molecule is an important regulator of T cell homeostasis and it has been linked to immune deficiencies as well as autoimmune disorders. Nevertheless, since Coronin 1A is strongly expressed in all leukocyte subsets, it is not conclusive whether or not this phenotype is attributed to a T cell-intrinsic function of Coronin 1A. To address this research question, we have generated a T cell-specific Coronin 1a knockout mouse (Coro1afl/fl x Cd4[Cre]). Deletion of Coro1a specifically in T cells led to a strong reduction in T cell number and a shift towards the effector/memory phenotype in peripheral lymphoid organs when compared to Cd4[Cre] mice expressing wild-type Coro1a. In contrast to peripheral lymphoid tissue, thymocyte number and subsets were not affected by the deletion of Coro1a. Furthermore, T cell-specific Coro1a knockout mice were largely resistant to the induction of autoimmunity when tested in the MOG-induced EAE mouse model of multiple sclerosis. Thus, the phenotype of T cell-specific Coro1a deletion resembles the phenotype observed with conventional (whole body) Coro1a knockout mice. In summary, our findings provide formal proof of the predominant T cell-intrinsic role of Coronin 1A.

Severe Molecular Defects Exhibited by the R179H Mutation in Human Vascular Smooth Muscle {alpha}-Actin [Protein Structure and Folding]

August 22nd, 2016 by Lu, H., Fagnant, P. M., Krementsova, E. B., Trybus, K. M.

Mutations in vascular smooth muscle α-actin (SM α-actin), encoded by ACTA2, are the most common cause of familial thoracic aortic aneurysms that lead to dissection (TAAD). The R179H mutation has a poor patient prognosis, and is unique in causing multisystemic smooth muscle dysfunction (1). Here we characterize this mutation in expressed human SM α-actin. R179H actin shows severe polymerization defects, with a 40-fold higher critical concentration for assembly than WT SM α-actin, driven by a high disassembly rate. The mutant filaments are more readily severed by cofilin. Both defects are attenuated by copolymerization with WT. The R179H monomer binds more tightly to profilin, and formin binding suppresses nucleation and slows polymerization rates. Linear filaments will thus not be readily formed, and cells expressing R179H actin will likely have increased levels of monomeric G-actin. The co-transcription factor myocardin-related transcription factor-A (MRTF-A), which affects cellular phenotype, binds R179H actin with less cooperativity than WT actin. Smooth muscle myosin moves R179H filaments more slowly than WT, even when copolymerized with equimolar amounts of WT. The marked decrease in the ability to form filaments may contribute to the poor patient prognosis, and explain why R179H disrupts even visceral smooth muscle cell function where the SM α-actin isoform is present in low amounts. The R179H mutation has the potential to affect actin structure and function in both the contractile domain of the cell, and the more dynamic cytoskeletal pool of actin, both of which are required for contraction.