A Pseudorabies Virus Serine/Threonine Kinase, US3, Promotes Retrograde Transport in Axons via Akt/mToRC1

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body. Importance Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

Impaired Retrograde Transport Due to Lack of TBC1D5 Contributes to the Trafficking Defect of Lysosomal Cathepsins in Ischemic/Hypoxic Cardiomyocytes

Lysosomal dysfunction has been found in many pathological conditions, and methods to improve lysosomal function have been reported to be protective against infarcted hearts. However, the mechanisms underlying lysosomal dysfunction caused by ischemic injury are far less well-established. The retromer complex is implicated in the trafficking of cation-independent mannose 6-phosphate receptor (CI-MPR), which is an important protein tag for the proper transport of lysosomal contents and therefore is important for the maintenance of lysosomal function. In this study, we found that the function of retrograde transport in cardiomyocytes was impaired with ischemia/hypoxia (I/H) treatment, which resulted in a decrease in CI-MPR and an abnormal distribution of lysosomal cathepsins. I/H treatment caused a reduction in TBC1D5 and a blockade of the Rab7 membrane cycle, which impeded retromer binding to microtubules and motor proteins, resulting in an impairment of retrograde transport and a decrease in CI-MPR. We also established that TBC1D5 was an important regulator of the distribution of lysosomal cathepsins. Our findings shed light on the regulatory role of retromer in ischemic injury and uncover the regulatory mechanism of TBC1D5 over retromer.

Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

Dynamic queuosine changes in tRNA couple nutrient levels to codon choice in Trypanosoma brucei

Every type of nucleic acid in cells undergoes programmed chemical post-transcriptional modification. Generally, modification enzymes use substrates derived from intracellular metabolism, one exception is queuine (q)/queuosine (Q), which eukaryotes obtain from their environment; made by bacteria and ultimately taken into eukaryotic cells via currently unknown transport systems. Here, we use a combination of molecular, cell biology and biophysical approaches to show that in Trypanosoma brucei tRNA Q levels change dynamically in response to concentration variations of a sub-set of amino acids in the growth media. Most significant were variations in tyrosine, which at low levels lead to increased Q content for all the natural tRNAs substrates of tRNA-guanine transglycosylase (TGT). Such increase results from longer nuclear dwell time aided by retrograde transport following cytoplasmic splicing. In turn high tyrosine levels lead to rapid decrease in Q content. Importantly, the dynamic changes in Q content of tRNAs have negligible effects on global translation or growth rate but, at least, in the case of tRNATyr it affected codon choice. These observations have implications for the occurrence of other tunable modifications important for ‘normal’ growth, while connecting the intracellular localization of modification enzymes, metabolites and tRNAs to codon selection and implicitly translational output.

FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

The retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.

A Novel Mutation c.841C>T in COPA Syndrome of an 11-Year-Old Boy: A Case Report and Short Literature Review

COPA syndrome is a rare autosomal dominant disorder with auto-immune and auto-inflammatory abnormalities. This disease is caused by mutations of COPα, a protein that functions in the retrograde transport from the Golgi to the ER. Here we report the first COPA case of an 11-year-old boy with c.841C>T, p.R281W mutation. The arginine at position 281 was located in a highly evolutionary-conserved region. Immunosuppressive drugs and corticosteroids might not improve the long-term outcome of COPA patients. For patients with pulmonary disease, polyarthritis and/or kidney disorder, and suspected of COPA, genetic analysis should be conducted promptly for early diagnosis.

New perspectives on cytoskeletal dysregulation and mitochondrial mislocalization in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective, early degeneration of motor neurons in the brain and spinal cord. Motor neurons have long axonal projections, which rely on the integrity of neuronal cytoskeleton and mitochondria to regulate energy requirements for maintaining axonal stability, anterograde and retrograde transport, and signaling between neurons. The formation of protein aggregates which contain cytoskeletal proteins, and mitochondrial dysfunction both have devastating effects on the function of neurons and are shared pathological features across several neurodegenerative conditions, including ALS, Alzheimer's disease, Parkinson's disease, Huntington’s disease and Charcot-Marie-Tooth disease. Furthermore, it is becoming increasingly clear that cytoskeletal integrity and mitochondrial function are intricately linked. Therefore, dysregulations of the cytoskeletal network and mitochondrial homeostasis and localization, may be common pathways in the initial steps of neurodegeneration. Here we review and discuss known contributors, including variants in genetic loci and aberrant protein activities, which modify cytoskeletal integrity, axonal transport and mitochondrial localization in ALS and have overlapping features with other neurodegenerative diseases. Additionally, we explore some emerging pathways that may contribute to this disruption in ALS.

The Chemical Genetic Interactions of Statin Drugs with Their Target Genes in Saccharomyces cerevisiae

<p>Statins, competitive inhibitors of the rate limiting cholesterol/ergosterol enzymes HMG-CoA reductase (HMG1 and HMG2), are the most widely prescribed human therapeutic drugs. They are effective in lowering cholesterol levels in atherosclerosis and related syndromes. However, statins exhibit a range of pleiotropic side effects whose mechanisms are poorly understood. This study investigates statin pleiotropy by analysis of genetic interaction networks in yeast, Saccharomyces cerevisiae, which shows high homology to mammalian pathways affected by statins. Synthetic genetic array (SGA) analysis allows elucidation of functional genetic networks of genes of interest ("query genes") by measurement of genetic epistasis in double mutants of the query gene with the genome - wide deletion mutant array of ~4800 non-essential strains. Chemicalgenetic profiling is similar where a SMP may effectively replace the query gene in genome wide epistatic analysis. The genetic interaction networks resulting from use of HMG1 and HMG2 as query genes for SGA analysis were compared to the chemical-genetic profiles of atorvastatin, cerivastatin and lovastatin. The genes ARV1, BTS1, OPI3 displaying phenotypic enhancements (i.e. their deletion caused major growth inhibition) with statins became essential in the presence of all the statins. Two mitochondrial genes, COX17 and MMM1, showed phenotypic suppressions (i.e. their deletion allowed better growth) in common to all three statin drugs. An attractive hypothesis is that major pleiotropic effects of statins could be due to variation in function or expression of these enhancing or suppressing genes. Other processes compensating statin use were also elucidated. For example, when HMG1 and its epistatically interacting genes are shut down by deletion coupled with inhibition of HMG2 with statin, there is strong evidence that the cell attempts to maintain membrane/lipid homeostasis via anterograde and retrograde transport mechanisms, including the mobilisation of lipid storage droplets. To aid refinement of genetic analysis in this and future studies, a more direct phenotypic assay was developed for quantifying ergosterol. Such an assay may be used as a phenotype to map the effect of up - and downstream - genes, or network genes affecting ergosterol levels. This assay was used to quantify ergosterol in a drug - resistant mutant developed by others aiding confirmation of the drug target.</p>

The Chemical Genetic Interactions of Statin Drugs with Their Target Genes in Saccharomyces cerevisiae

<p>Statins, competitive inhibitors of the rate limiting cholesterol/ergosterol enzymes HMG-CoA reductase (HMG1 and HMG2), are the most widely prescribed human therapeutic drugs. They are effective in lowering cholesterol levels in atherosclerosis and related syndromes. However, statins exhibit a range of pleiotropic side effects whose mechanisms are poorly understood. This study investigates statin pleiotropy by analysis of genetic interaction networks in yeast, Saccharomyces cerevisiae, which shows high homology to mammalian pathways affected by statins. Synthetic genetic array (SGA) analysis allows elucidation of functional genetic networks of genes of interest ("query genes") by measurement of genetic epistasis in double mutants of the query gene with the genome - wide deletion mutant array of ~4800 non-essential strains. Chemicalgenetic profiling is similar where a SMP may effectively replace the query gene in genome wide epistatic analysis. The genetic interaction networks resulting from use of HMG1 and HMG2 as query genes for SGA analysis were compared to the chemical-genetic profiles of atorvastatin, cerivastatin and lovastatin. The genes ARV1, BTS1, OPI3 displaying phenotypic enhancements (i.e. their deletion caused major growth inhibition) with statins became essential in the presence of all the statins. Two mitochondrial genes, COX17 and MMM1, showed phenotypic suppressions (i.e. their deletion allowed better growth) in common to all three statin drugs. An attractive hypothesis is that major pleiotropic effects of statins could be due to variation in function or expression of these enhancing or suppressing genes. Other processes compensating statin use were also elucidated. For example, when HMG1 and its epistatically interacting genes are shut down by deletion coupled with inhibition of HMG2 with statin, there is strong evidence that the cell attempts to maintain membrane/lipid homeostasis via anterograde and retrograde transport mechanisms, including the mobilisation of lipid storage droplets. To aid refinement of genetic analysis in this and future studies, a more direct phenotypic assay was developed for quantifying ergosterol. Such an assay may be used as a phenotype to map the effect of up - and downstream - genes, or network genes affecting ergosterol levels. This assay was used to quantify ergosterol in a drug - resistant mutant developed by others aiding confirmation of the drug target.</p>

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.