The choroid plexus links innate immunity to CSF dysregulation in hydrocephalus.

The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Dual regulation of SLC25A39 by AFG3L2 and iron controls mitochondrial glutathione homeostasis.

Organelle transporters define metabolic compartmentalization, and how this metabolite transport process can be modulated is poorly explored. Here, we discovered that human SLC25A39, a mitochondrial transporter critical for mitochondrial glutathione uptake, is a short-lived protein under dual regulation at the protein level. Co-immunoprecipitation mass spectrometry and CRISPR knockout (KO) in mammalian cells identified that mitochondrial m-AAA protease AFG3L2 is responsible for degrading SLC25A39 through the matrix loop 1. SLC25A39 senses mitochondrial iron-sulfur cluster using four matrix cysteine residues and inhibits its degradation. SLC25A39 protein regulation is robust in developing and mature neurons. This dual transporter regulation, by protein quality control and metabolic sensing, allows modulating mitochondrial glutathione level in response to iron homeostasis, opening avenues for exploring regulation of metabolic compartmentalization. Neuronal SLC25A39 regulation connects mitochondrial protein quality control, glutathione, and iron homeostasis, which were previously unrelated biochemical features in neurodegeneration.

Phosphorylation of the nuclear poly(A) binding protein (PABPN1) during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.

Widespread association of the Argonaute protein AGO2 with meiotic chromatin suggests a distinct nuclear function in mammalian male reproduction.

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

DOT1L bridges transcription and heterochromatin formation at mammalian pericentromeres.

Repetitive DNA elements are packaged in heterochromatin, but many require bursts of transcription to initiate and maintain long-term silencing. The mechanisms by which these heterochromatic genome features are transcribed remain largely unknown. Here, we show that DOT1L, a conserved histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), has a specialized role in transcription of major satellite repeats to maintain pericentromeric heterochromatin and genome stability. We find that H3K79me3 is selectively enriched relative to H3K79me2 at repetitive elements in mouse embryonic stem cells (mESCs), that DOT1L loss compromises pericentromeric satellite transcription, and that this activity involves possible coordination between DOT1L and the chromatin remodeler SMARCA5. Stimulation of transcript production from pericentromeric repeats by DOT1L participates in stabilization of heterochromatin structures in mESCs and cleavage-stage embryos and is required for preimplantation viability. Our findings uncover an important role for DOT1L as a bridge between transcriptional activation of repeat elements and heterochromatin stability, advancing our understanding of how genome integrity is maintained and how chromatin state is set up during early development.

Diversification of a Salmonella virulence protein function by ubiquitin-dependent differential localization.

Many bacterial pathogens and symbionts utilize type III secretion systems to deliver bacterial effector proteins into host cells. These effector proteins have the capacity to modulate a large variety of cellular functions in a highly regulated manner. Here, we report that the phosphoinositide phosphatase SopB, a Salmonella Typhimurium type III secreted effector protein, diversifies its function by localizing to different cellular compartments in a ubiquitin-dependent manner. We show that SopB utilizes the same enzymatic activity to modulate actin-mediated bacterial internalization and Akt activation at the plasma membrane and vesicular trafficking and intracellular bacterial replication at the phagosome. Thus, by exploiting the host cellular machinery, Salmonella Typhimurium has evolved the capacity to broaden the functional repertoire of a virulence factor to maximize its ability to modulate cellular functions.

Differential Effects of Cocaine and Morphine on the Diurnal Regulation of the Mouse Nucleus Accumbens Proteome.

Substance use disorders are associated with disruptions in sleep and circadian rhythms that persist during abstinence and may contribute to relapse risk. Repeated use of substances such as psychostimulants and opioids may lead to significant alterations in molecular rhythms in the nucleus accumbens (NAc), a brain region central to reward and motivation. Previous studies have identified rhythm alterations in the transcriptome of the NAc and other brain regions following the administration of psychostimulants or opioids. However, little is known about the impact of substance use on the diurnal rhythms of the proteome in the NAc. We used liquid chromatography coupled to tandem mass spectrometry-based quantitative proteomics, along with a data-independent acquisition analysis pipeline, to investigate the effects of cocaine or morphine administration on diurnal rhythms of proteome in the mouse NAc. Overall, our data reveal cocaine and morphine differentially alter diurnal rhythms of the proteome in the NAc, with largely independent differentially expressed proteins dependent on time-of-day. Pathways enriched from cocaine altered protein rhythms were primarily associated with glucocorticoid signaling and metabolism, whereas morphine was associated with neuroinflammation. Collectively, these findings are the first to characterize the diurnal regulation of the NAc proteome and demonstrate a novel relationship between the phase-dependent regulation of protein expression and the differential effects of cocaine and morphine on the NAc proteome. The proteomics data in this study are available via ProteomeXchange with identifier PXD042043.

Autoinhibition of the GEF activity of cytoskeletal regulatory protein Trio is disrupted in neurodevelopmental disorder-related genetic variants.

TRIO encodes a cytoskeletal regulatory protein with three catalytic domains-two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain-as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both in vitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.

Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids.

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A2, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (Vmax∼15.5 µmol/min/mg; Km∼83 µM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall-deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.

Liver-Dependent Lung Remodeling during Systemic Inflammation Shapes Responses to Secondary Infection.

Systemic duress, such as that elicited by sepsis, burns, or trauma, predisposes patients to secondary pneumonia, demanding better understanding of host pathways influencing this deleterious connection. These pre-existing circumstances are capable of triggering the hepatic acute-phase response (APR), which we previously demonstrated is essential for limiting susceptibility to secondary lung infections. To identify potential mechanisms underlying protection afforded by the lung-liver axis, our studies aimed to evaluate liver-dependent lung reprogramming when a systemic inflammatory challenge precedes pneumonia. Wild-type mice and APR-deficient littermate mice with hepatocyte-specific deletion of STAT3 (hepSTAT3-/-), a transcription factor necessary for full APR initiation, were challenged i.p. with LPS to induce endotoxemia. After 18 h, pneumonia was induced by intratracheal Escherichia coli instillation. Endotoxemia elicited significant transcriptional alterations in the lungs of wild-type and hepSTAT3-/- mice, with nearly 2000 differentially expressed genes between genotypes. The gene signatures revealed exaggerated immune activity in the lungs of hepSTAT3-/- mice, which were compromised in their capacity to launch additional cytokine responses to secondary infection. Proteomics revealed substantial liver-dependent modifications in the airspaces of pneumonic mice, implicating a network of dispatched liver-derived mediators influencing lung homeostasis. These results indicate that after systemic inflammation, liver acute-phase changes dramatically remodel the lungs, resulting in a modified landscape for any stimuli encountered thereafter. Based on the established vulnerability of hepSTAT3-/- mice to secondary lung infections, we believe that intact liver function is critical for maintaining the immunological responsiveness of the lungs.

Small Extracellular Vesicles Control Dendritic Spine Development through Regulation of HDAC2 Signaling.

The release of small extracellular vesicles (sEVs) has recently been reported, but knowledge of their function in neuron development remains limited. Using LC-MS/MS, we found that sEVs released from developing cortical neurons in vitro obtained from mice of both sexes were enriched in cytoplasm, exosome, and protein-binding and DNA/RNA-binding pathways. The latter included HDAC2, which was of particular interest, because HDAC2 regulates spine development, and populations of neurons expressing different levels of HDAC2 co-exist in vivo during the period of spine growth. Here, we found that HDAC2 levels decrease in neurons as they acquire synapses and that sEVs from HDAC2-rich neurons regulate HDAC2 signaling in HDAC2-low neurons possibly through HDAC2 transfer. This regulation led to a transcriptional decrease in HDAC2 synaptic targets and the density of excitatory synapses. These data suggest that sEVs provide inductive cell-cell signaling that coordinates the development of dendritic spines via the activation of HDAC2-dependent transcriptional programs.SIGNIFICANCE STATEMENT A role of small extracellular vesicles (sEVs; also called exosomes) in neuronal development is of particular interest, because sEVs could provide a major signaling modality between developing neurons when synapses are not fully functional or immature. However, knowledge of sEVs on neuron, and more precisely spine development, is limited. We provide several lines of evidence that sEVs released from developing cortical neurons regulate the development of dendritic spines via the regulation of HDAC2 signaling. This paracrine communication is temporally restricted during development because of the age-dependent decrease in sEV release as neurons mature and acquire spines.

Granulocyte-Colony Stimulating Factor Reduces Cocaine-Seeking and Downregulates Glutamatergic Synaptic Proteins in Medial Prefrontal Cortex.

Psychostimulant use disorder is a major public health issue, and despite the scope of the problem there are currently no Food and Drug Administration (FDA)-approved treatments. There would be tremendous utility in development of a treatment that could help patients both achieve and maintain abstinence. Previous work from our group has identified granulocyte-colony stimulating factor (G-CSF) as a neuroactive cytokine that alters behavioral response to cocaine, increases synaptic dopamine release, and enhances cognitive flexibility. Here, we investigate the role of G-CSF in affecting extinction and reinstatement of cocaine-seeking and perform detailed characterization of its proteomic effects in multiple limbic substructures. Male Sprague Dawley rats were injected with PBS or G-CSF during (1) extinction or (2) abstinence from cocaine self-administration, and drug seeking behavior was measured. Quantitative assessment of changes in the proteomic landscape in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were performed via data-independent acquisition (DIA) mass spectrometry analysis. Administration of G-CSF during extinction accelerated the rate of extinction, and administration during abstinence attenuated cue-induced cocaine-seeking. Analysis of global protein expression demonstrated that G-CSF regulated proteins primarily in mPFC that are critical to glutamate signaling and synapse maintenance. Taken together, these findings support G-CSF as a viable translational research target with the potential to reduce drug craving or seeking behaviors. Importantly, recombinant G-CSF exists as an FDA-approved medication which may facilitate rapid clinical translation. Additionally, using cutting-edge multiregion discovery proteomics analyses, these studies identify a novel mechanism underlying G-CSF effects on behavioral plasticity.SIGNIFICANCE STATEMENT Pharmacological treatments for psychostimulant use disorder are desperately needed, especially given the disease's chronic, relapsing nature. However, there are currently no Food and Drug Administration (FDA)-approved pharmacotherapies. Emerging evidence suggests that targeting the immune system may be a viable translational research strategy; preclinical studies have found that the neuroactive cytokine granulocyte-colony stimulating factor (G-CSF) alters cocaine reward and reinforcement and can enhance cognitive flexibility. Given this basis of evidence we studied the effects of G-CSF treatment on extinction and reinstatement of cocaine seeking. We find that administration of G-CSF accelerates extinction and reduces cue-induced drug seeking after cocaine self-administration. In addition, G-CSF leads to downregulation of synaptic glutamatergic proteins in medial prefrontal cortex (mPFC), suggesting that G-CSF influences drug seeking via glutamatergic mechanisms.

Early onset of senescence and imbalanced epidermal homeostasis across the decades in photoexposed human skin: Fingerprints of inflammaging.

Inflammaging is a theory of ageing which purports that low-level chronic inflammation leads to cellular dysfunction and premature ageing of surrounding tissue. Skin is susceptible to inflammaging because it is the first line of defence from the environment, particularly solar radiation. To better understand the impact of ageing and photoexposure on epidermal biology, we performed a system biology-based analysis of photoexposed face and arm, and photoprotected buttock sites, from women between the ages of 20s to 70s. Biopsies were analysed by histology, transcriptomics, and proteomics and skin surface biomarkers collected from tape strips. We identified morphological changes with age of epidermal thinning, rete ridge pathlength loss and stratum corneum thickening. The SASP biomarkers IL-8 and IL-1RA/IL1-α were consistently elevated in face across age and cis/trans-urocanic acid were elevated in arms and face with age. In older arms, the DNA damage response biomarker 53BP1 showed higher puncti numbers in basal layers and epigenetic ageing were accelerated. Genes associated with differentiation and senescence showed increasing expression in the 30s whereas genes associated with hypoxia and glycolysis increased in the 50's. Proteomics comparing 60's vs 20's confirmed elevated levels of differentiation and glycolytic-related proteins. Representative immunostaining for proteins of differentiation, senescence and oxygen sensing/hypoxia showed similar relationships. This system biology-based analysis provides a body of evidence that young photoexposed skin is undergoing inflammaging. We propose the presence of chronic inflammation in young skin contributes to an imbalance of epidermal homeostasis that leads to a prematurely aged appearance during later life.

A single phosphoacceptor residue in BGLF3 is essential for transcription of Epstein-Barr virus late genes.

Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses.

Glucocorticoids and serum- and glucocorticoid-inducible kinase 1 are potent regulators of CFTR in the native intestine: implications for stress-induced diarrhea.

Nongenomic glucocorticoid (GC) and serum- and glucocorticoid-inducible kinase 1 (SGK1) signaling regulate ion transport, but CFTR has not been investigated in the intestine. We examined GC, SGK1, and phosphatidylinositol 3-kinase (PI3K) kinase signaling of CFTR ion transport in native intestine and the role of GCs on mRNA, protein, surface expression, and cyclic guanosine monophosphate (cGMP)-elicited diarrhea. Rats were treated with dexamethasone (DEXA; 2 mg/kg ip) or DMSO for 1, 4, and 24 h. Cyclic adenosine monophosphate (cAMP)-activated ion transport was examined in the presence or absence of SGK1 and PI3K inhibitors. Phosphorylation of SGK1, phosphoinositide-dependent kinase 1, and Akt kinases was confirmed by immunoblots using phosphor-specific antibodies. Tissue lysates were analyzed by mass spectrometry. CFTR and SGK1 mRNA were measured by quantitative PCR. Changes in total and surface CFTR protein were determined. The role of GC in cGMP-activated CFTR ion transport was examined. GC synergistically increased CFTR ion transport by SGK1 and PI3K signaling and increased CFTR protein without altering SGK1 or CFTR mRNA. GC induced highest levels of CFTR protein at 4 h that were associated with marked increase in surface CFTR, phosphorylation of the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-like (Nedd4-2), and 14-3-3ε, supporting their roles in surface retention and stability. Coimmunoprecipitation of CFTR, Nedd4-2, and 14-3-3ε indicated that assembly of this complex is a likely effector of the SGK and Akt pathways. Mass spectrometry identified phosphorylated peptides in relevant proteins. GC-SGK1 potently regulates CFTR in the intestine and is implicated in diarrheal disease.NEW & NOTEWORTHY This is the first study to examine the mechanisms of glucocorticoid, serum- and glucocorticoid-inducible kinase 1, and nongenomic kinase signaling of CFTR in the native intestine. We identified unique and druggable intestine-specific factors of the pathway that are targets for treating stress-induced diarrhea.

ProteomicsBrowser: MS/proteomics data visualization and investigation.

SUMMARY: Large-scale, quantitative proteomics data are being generated at ever increasing rates by high-throughput, mass spectrometry technologies. However, due to the complexity of these large datasets as well as the increasing numbers of post-translational modifications (PTMs) that are being identified, developing effective methods for proteomic visualization has been challenging. ProteomicsBrowser was designed to meet this need for comprehensive data visualization. Using peptide information files exported from mass spectrometry search engines or quantitative tools as input, the peptide sequences are aligned to an internal protein database such as UniProtKB. Each identified peptide ion including those with PTMs is then visualized along the parent protein in the Browser. A unique property of ProteomicsBrowser is the ability to combine overlapping peptides in different ways to focus analysis of sequence coverage, charge state or PTMs. ProteomicsBrowser includes other useful functions, such as a data filtering tool and basic statistical analyses to qualify quantitative data. AVAILABILITY AND IMPLEMENTATION: ProteomicsBrowser is implemented in Java8 and is available at https://medicine.yale.edu/keck/nida/proteomicsbrowser.aspx and https://github.com/peng-gang/ProteomicsBrowser. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cerebrospinal fluid proteome evaluation in major depressive disorder by mass spectrometry.

BACKGROUND: Depression affects approximately 7.1% of the United States population every year and has an annual economic burden of over $210 billion dollars. Several recent studies have sought to investigate the pathophysiology of depression utilizing focused cerebrospinal fluid (CSF) and serum analysis. Inflammation and metabolic dysfunction have emerged as potential etiological factors from these studies. A dysregulation in the levels of inflammatory proteins such as IL-12, TNF, IL-6 and IFN-γ have been found to be significantly correlated with depression. METHODS: CSF samples were obtained from 15 patients, seven with major depressive disorder and eight age- and gender-matched non-psychiatric controls. CSF protein profiles were obtained using quantitative mass spectrometry. The data were analyzed by Progenesis QI proteomics software to identify significantly dysregulated proteins. The results were subjected to bioinformatics analysis using the Ingenuity Pathway Analysis suite to obtain unbiased mechanistic insight into biologically relevant interactions and pathways. RESULTS: Several dysregulated proteins were identified. Bioinformatics analysis indicated that the potential disorder/disease pathways include inflammatory response, metabolic disease and organismal injury. Molecular and cellular functions that were affected include cellular compromise, cell-to-cell signaling & interaction, cellular movement, protein synthesis, and cellular development. The major canonical pathway that was upregulated was acute phase response signaling. Endogenous upstream regulators that may influence dysregulation of proinflammatory molecules associated with depression are interleukin-6 (IL-6), signal transducer and activator of transcription 3 (STAT3), oncostatin M, PR domain zinc finger protein 1 (PRDM1), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). CONCLUSIONS: The proteome profiling data in this report identifies several potential biological functions that may be involved in the pathophysiology of major depressive disorder. Future research into how the differential expression of these proteins is involved in the etiology and severity of depression will be important.

Phosphorylation of human placental aromatase CYP19A1.

Aromatase CYP19A1 catalyzes the synthesis of estrogens in endocrine, reproductive and central nervous systems. Higher levels of 17ß-estradiol (E2) are associated with malignancies and diseases of the breast, ovary and endometrium, while low E2 levels increase the risk for osteoporosis, cardiovascular diseases and cognitive disorders. E2, the transcriptional activator of the estrogen receptors, is also known to be involved in non-genomic signaling as a neurotransmitter/neuromodulator, with recent evidence for rapid estrogen synthesis (RES) within the synaptic terminal. Although regulation of brain aromatase activity by phosphorylation/dephosphorylation has been suggested, it remains obscure in the endocrine and reproductive systems. RES and overabundance of estrogens could stimulate the genomic and non-genomic signaling pathways, and genotoxic effects of estrogen metabolites. Here, by utilizing biochemical, cellular, mass spectrometric, and structural data we unequivocally demonstrate phosphorylation of human placental aromatase and regulation of its activity. We report that human aromatase has multiple phosphorylation sites, some of which are consistently detectable. Phosphorylation of the residue Y361 at the reductase-coupling interface significantly elevates aromatase activity. Other sites include the active site residue S478 and several at the membrane interface. We present the evidence that two histidine residues are phosphorylated. Furthermore, oxidation of two proline residues near the active site may have implications in regulation. Taken together, the results demonstrate that aromatase activity is regulated by phosphorylation and possibly other post-translational modifications. Protein level regulation of aromatase activity not only represents a paradigm shift in estrogen-mediated biology, it could also explain unresolved clinical questions such as aromatase inhibitor resistance.

Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.