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Air pollution consists of complex mixtures of chemicals with serious deleterious health effects from acute and chronic exposure. To help understand the mechanisms by which adverse effects occur, the present work examines the responses of cultured human epidermal keratinocytes to specific chemicals commonly found in woodsmoke. Our earlier findings with liquid smoke flavoring (aqueous extract of charred wood) revealed that such extracts stimulated the expression of genes associated with oxidative stress and proinflammatory response, activated the aryl hydrocarbon receptor, thereby inducing cytochrome P4501A1 activity, and induced cross-linked envelope formation, a lethal event ordinarily occurring during terminal differentiation. The present results showed that furfural produced transcriptional responses resembling those of liquid smoke, cyclohexanedione activated the aryl hydrocarbon receptor, and several chemicals induced envelope formation. Of these, syringol permeabilized the cells to the egress of lactate dehydrogenase at a concentration close to that yielding envelope formation, while furfural induced envelope formation without permeabilization detectable in this way. Furfural (but not syringol) stimulated the incorporation of amines into cell proteins in extracts in the absence of transglutaminase activity. Nevertheless, both chemicals substantially increased the amount of cellular protein incorporated into envelopes and greatly altered the envelope protein profile. Moreover, the proportion of keratin in the envelopes was dramatically increased. These findings are consistent with the chemically induced protein cross-linking in the cells. Elucidating mechanisms by which this phenomenon occurs may help understand how smoke chemicals interact with proteins to elicit cellular responses, interpret bioassays of complex pollutant mixtures, and suggest additional sensitive ways to monitor exposures.

Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

The fetal brain of the mouse is thought to be dependent upon the placenta as a source of serotonin (5-hydroxytryptamine; 5-HT) and other factors. How factors reach the developing brain remains uncertain but are postulated here to be part of the cargo carried by placental extracellular vesicles (EV). We have analyzed the protein, catecholamine, and small RNA content of EV from mouse trophoblast stem cells (TSC) and TSC differentiated into parietal trophoblast giant cells (pTGC), potential primary purveyors of 5-HT. Current studies examined how exposure of mouse neural progenitor cells (NPC) to EV from either TSC or pTGC affect their transcriptome profiles. The EV from trophoblast cells contained relatively high amounts of 5-HT, as well as dopamine and norepinephrine, but there were no significant differences between EV derived from pTGC and from TSC. Content of miRNA and small nucleolar (sno)RNA, however, did differ according to EV source, and snoRNA were upregulated in EV from pTGC. The primary inferred targets of the microRNA (miRNA) from both pTGC and TSC were mRNA enriched in the fetal brain. NPC readily internalized EV, leading to changes in their transcriptome profiles. Transcripts regulated were mainly ones enriched in neural tissues. The transcripts in EV-treated NPC that demonstrated a likely complementarity with miRNA in EV were mainly up- rather than downregulated, with functions linked to neuronal processes. Our results are consistent with placenta-derived EV providing direct support for fetal brain development and being an integral part of the placenta-brain axis.

ATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes encompassing canonical autophagy and noncanonical processes. Remarkably, genetic ablation of ATG5 in myeloid cells leads to an exacerbated pathological state in murine models of tuberculosis, characterized by an early surge in mortality much more severe when compared to the depletion of other components involved in Atg8ylation or canonical autophagy. This study shows that in the absence of ATG5, but not other core canonical autophagy factors, endolysosomal organelles display a lysosomal hypersensitivity phenotype when subjected to damage. This is in part due to a compromised recruitment of ESCRT proteins to lysosomes in need of repair. Mechanistically, in the absence of ATG5, the ESCRT protein PDCD6IP/ALIX is sequestered by the alternative conjugate ATG12-ATG3, contributing to excessive exocytic processes while not being available for lysosomal repair. Specifically, this condition increases secretion of extracellular vesicles and particles, and leads to excessive degranulation in neutrophils. Our findings uncover unique functions of ATG5 outside of the autophagy and Atg8ylation paradigm. This finding is of in vivo relevance for tuberculosis pathogenesis as modeled in mice.Abbreviations: Atg5: autophagy related 5; ESCRT: endosomal sorting complex required for transport; EVPs: extracellular vesicles and particles; FPR1: formyl peptide receptor 1; LyHYP: lysosomal hypersensitivity phenotype; LysoIP: lysosome immunopurification; Mtb: Mycobacterium tuberculosis; ORF3a: open reading frame 3a protein; PDCD6IP/ALIX: programmed cell death 6 interacting protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, TFEB: transcription factor EB.

Alcohol-associated liver disease (ALD) is a prevalent medical problem with limited effective treatment strategies. Although many biological processes contributing to ALD have been elucidated, a complete understanding of the underlying mechanisms is still lacking. The current study employed a proteomic approach to identify hepatic changes resulting from ethanol (EtOH) consumption and the genetic ablation of the formyl peptide receptor 2 (FPR2), a G-protein coupled receptor known to regulate multiple signaling pathways and biological processes, in a mouse model of ALD. Since previous research from our team demonstrated a notable reduction in hepatic FPR2 protein levels in patients with alcohol-associated hepatitis (AH), the proteomic changes in the livers of Fpr2-/- EtOH mice were compared to those observed in patients with AH in order to identify common hepatic proteomic alterations. Several pathways linked to exacerbated ALD in Fpr2-/- EtOH mice, as well as hepatic protein changes resembling those found in patients suffering from AH, were identified. These alterations included decreased levels of coagulation factors F2 and F9, as well as reduced hepatic levels of glutamate-cysteine ligase catalytic subunit (GCLC) and total glutathione in Fpr2-/- EtOH compared to WT EtOH mice. In conclusion, the data suggest that FPR2 may play a regulatory role in hepatic blood coagulation and the antioxidant system, both in a pre-clinical model of ALD and in human AH, however further experiments are required to validate these findings.

N-Myc is a key driver of neuroblastoma and neuroendocrine prostate cancer (NEPC). One potential way to circumvent the challenge of undruggable N-Myc is to target the protein homeostasis (proteostasis) system that maintains N-Myc levels. Here, we identify heat shock protein 70 (HSP70) as a top partner of N-Myc, which binds a conserved "SELILKR" motif and prevents the access of E3 ubiquitin ligase, STIP1 homology and U-box containing protein 1 (STUB1), possibly through steric hindrance. When HSP70's dwell time on N-Myc is increased by treatment with the HSP70 allosteric inhibitor, STUB1 is in close proximity with N-Myc and becomes functional to promote N-Myc ubiquitination on the K416 and K419 sites and forms polyubiquitination chains linked by the K11 and K63 sites. Notably, HSP70 inhibition significantly suppressed NEPC tumor growth, increased the efficacy of aurora kinase A (AURKA) inhibitors, and limited the expression of neuroendocrine-related pathways.

[This corrects the article DOI: 10.1371/journal.pone.0290778.].

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

Despite the advantages of fewer missing values by collecting fragment ion data on all analytes in the sample as well as the potential for deeper coverage, the adoption of data-independent acquisition (DIA) in proteomics core facility settings has been slow. The Association of Biomolecular Resource Facilities conducted a large interlaboratory study to evaluate DIA performance in proteomics laboratories with various instrumentation. Participants were supplied with generic methods and a uniform set of test samples. The resulting 49 DIA datasets act as benchmarks and have utility in education and tool development. The sample set consisted of a tryptic HeLa digest spiked with high or low levels of 4 exogenous proteins. Data are available in MassIVE MSV000086479. Additionally, we demonstrate how the data can be analyzed by focusing on 2 datasets using different library approaches and show the utility of select summary statistics. These data can be used by DIA newcomers, software developers, or DIA experts evaluating performance with different platforms, acquisition settings, and skill levels.

The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage.Abbreviations: Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame 3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1: TIA1 cytotoxic granule associated RNA binding protein.

ATG5 is a part of the E3 ligase directing lipidation of ATG8 proteins, a process central to membrane atg8ylation and canonical autophagy. Loss of Atg5 in myeloid cells causes early mortality in murine models of tuberculosis. This in vivo phenotype is specific to ATG5. Here, we show using human cell lines that absence of ATG5, but not of other ATGs directing canonical autophagy, promotes lysosomal exocytosis and secretion of extracellular vesicles and, in murine Atg5fl/fl LysM-Cre neutrophils, their excessive degranulation. This is due to lysosomal disrepair in ATG5 knockout cells and the sequestration by an alternative conjugation complex, ATG12-ATG3, of ESCRT protein ALIX, which acts in membrane repair and exosome secretion. These findings reveal a previously undescribed function of ATG5 in its host-protective role in murine experimental models of tuberculosis and emphasize the significance of the branching aspects of the atg8ylation conjugation cascade beyond the canonical autophagy.

PurposeFuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease that impacts the structure and stiffness of the Descemet's membrane (DM), the substratum for corneal endothelial cells (CECs). These structural alterations of the DM could contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and transforming growth factor-β (TGF-β) pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid (AA) is found at high concentrations in FECD and its impact on CEC survival has been investigated. However, how TGF-β and AA effect the composition and rigidity of the CEC's matrix remains unknown.MethodsIn this study, we investigated the effect of AA, TGF-β1 and TGF-β3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix (ECM) secreted by primary bovine corneal endothelial cells (BCECs).ResultsImmunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-β1 and TGF-β3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-β induces the expression of matrix proteins collagen IV, laminin, and lysyl oxidase homolog 1.ConclusionsMolecular pathways identified in this study demonstrate the differential role of soluble factors in the pathogenesis of FECD.

Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. Significant traces of prometryn are documented in the environment, mainly in waters, soil, and plants used for human and domestic consumption. Previous studies have shown that triazine herbicides have carcinogenic potential in humans. However, there is limited information about the effects of prometryn on the cardiac system in the literature, or the mechanisms and signaling pathways underlying any potential cytotoxic effects are not known. It is important to understand the possible effects of exogenous compounds such as prometryn on the heart. To determine the mechanisms and signaling pathways affected by prometryn (185 mg/kg every 48 h for seven days), we performed proteomic profiling of male mice heart with quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. The data suggest that several major pathways, including energy metabolism, protein degradation, fatty acid metabolism, calcium signaling, and antioxidant defense system were altered in the hearts of prometryn-treated mice. Proteasome and immunoproteasome activity assays and expression levels showed proteasome dysfunction in the hearts of prometryn-treated mice. The results suggest that prometryn induced changes in mitochondrial function and various signaling pathways within the heart, particularly affecting stress-related responses.

Neonates have different cellular composition in their bronchoalveolar lavage fluid (BALF) when compared to foals and adult horses; however, little is known about the non-cellular components of BALF. The objective of this study was to determine the proteomic composition of BALF in neonatal horses and to compare it to that of foals and adult horses. Bronchoalveolar lavage fluid samples of seven neonates (< 1 week age), four 5 to 7-week-old foals, and six adult horses were collected. Quantitative proteomics of the fluid was performed using tandem mass tag labeling followed by high resolution liquid chromatography tandem mass spectrometry and protein relative abundances were compared between groups using exact text. A total of 704 proteins were identified with gene ontology terms and were classified. Of these, 332 proteins were related to the immune system in neonates, foals, and adult horses. The most frequent molecular functions identified were binding and catalytic activity and the most common biological processes were cellular process, metabolic process, and biological regulation. There was a significant difference in the proteome of neonates when compared to foals and to adult horses. Neonates had less relative expression (FDR < 0.01) of many immune-related proteins, including immunoglobulins, proteins involved in the complement cascade, ferritin, BPI fold-containing family B member 1, and macrophage receptor MARCO. This is the first report of equine neonate BALF proteomics and reveals differential abundance of proteins when compared to BALF from adult horses. The lower relative abundance of immune-related proteins in neonates could contribute to their susceptibility to pulmonary infections.

Protein profiling offers an effective approach to characterizing how far epidermis departs from normal in disease states. The present pilot investigation tested the hypothesis that protein expression in epidermal corneocytes is perturbed in the forehead of subjects exhibiting frontal fibrosing alopecia. To this end, samples were collected by tape stripping from subjects diagnosed with this condition and compared to those from asymptomatic control subjects and from those exhibiting androgenetic alopecia. Unlike the latter, which exhibited only 3 proteins significantly different from controls in expression level, forehead samples from frontal fibrosing alopecia subjects displayed 72 proteins significantly different from controls, nearly two-thirds having lower expression. The results demonstrate frontal fibrosing alopecia exhibits altered corneocyte protein expression in epidermis beyond the scalp, indicative of a systemic condition. They also provide a basis for quantitative measures of departure from normal by assaying forehead epidermis, useful in monitoring response to treatment while avoiding invasive biopsy.

The most common inherited cause of two genetically and clinico-pathologically overlapping neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is the presence of expanded GGGGCC intronic hexanucleotide repeats in the C9orf72 gene. Aside from haploinsufficiency and toxic RNA foci, another non-exclusive disease mechanism is the non-canonical translation of the repeat RNA into five different dipeptide repeat proteins (DPRs), which form neuronal inclusions in affected patient brains. While evidence from cellular and animal models supports a toxic gain-of-function of pathologic poly-GA, poly-GR, and poly-PR aggregates in promoting deposition of TDP-43 pathology and neurodegeneration in affected brain areas, the relative contribution of DPRs to the disease process in c9FTD/ALS patients remains unclear. Here we have used the proximity-dependent biotin identification (BioID) proximity proteomics approach to investigate the formation and collective composition of DPR aggregates using cellular models. While interactomes of arginine rich poly-GR and poly-PR aggregates overlapped and were enriched for nucleolar and ribosomal proteins, poly-GA aggregates demonstrated a distinct association with proteasomal components, molecular chaperones (HSPA1A/HSP70, HSPA8/HSC70, VCP/p97), co-chaperones (BAG3, DNAJA1A) and other factors that regulate protein folding and degradation (SQSTM1/p62, CALR, CHIP/STUB1). Experiments in cellular models of poly-GA pathology show that molecular chaperones and co-chaperones are sequestered to the periphery of dense cytoplasmic aggregates, causing depletion from their typical cellular localization. Their involvement in the pathologic process is confirmed in autopsy brain tissue, where HSPA8, BAG3, VCP, and its adapter protein UBXN6 show a close association with poly-GA aggregates in the frontal cortex, temporal cortex, and hippocampus of c9FTLD and c9ALS cases. The association of heat shock proteins and co-chaperones with poly-GA led us to investigate their potential role in reducing its aggregation. We identified HSP40 co-chaperones of the DNAJB family as potent modifiers that increased the solubility of poly-GA, highlighting a possible novel therapeutic avenue and a central role of molecular chaperones in the pathogenesis of human C9orf72-linked diseases.

We report that lysosomal damage is a hitherto unknown inducer of stress granule (SG) formation and that the process termed membrane atg8ylation coordinates SG formation with mTOR inactivation during lysosomal stress. SGs were induced by lysosome-damaging agents including SARS-CoV-2ORF3a, Mycobacterium tuberculosis, and proteopathic tau. During damage, mammalian ATG8s directly interacted with the core SG proteins NUFIP2 and G3BP1. Atg8ylation was needed for their recruitment to damaged lysosomes independently of SG condensates whereupon NUFIP2 contributed to mTOR inactivation via the Ragulator-RagA/B complex. Thus, cells employ membrane atg8ylation to control and coordinate SG and mTOR responses to lysosomal damage.

Mass spectrometry (MS) based diagnostic detection of 2019 novel coronavirus infectious disease (COVID-19) has been postulated to be a useful alternative to classical PCR based diagnostics. These MS based approaches have the potential to be both rapid and sensitive and can be done on-site without requiring a dedicated laboratory or depending on constrained supply chains (i.e., reagents and consumables). Matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS has a long and established history of microorganism detection and systemic disease assessment. Previously, we have shown that automated machine learning (ML) enhanced MALDI-TOF-MS screening of nasal swabs can be both sensitive and specific for COVID-19 detection. The underlying molecules responsible for this detection are generally unknown nor are they required for this automated ML platform to detect COVID-19. However, the identification of these molecules is important for understanding both the mechanism of detection and potentially the biology of the underlying infection. Here, we used nanoscale liquid chromatography tandem MS to identify endogenous peptides found in nasal swab saline transport media to identify peptides in the same the mass over charge (m/z) values observed by the MALDI-TOF-MS method. With our peptidomics workflow, we demonstrate that we can identify endogenous peptides and endogenous protease cut sites. Further, we show that SARS-CoV-2 viral peptides were not readily detected and are highly unlikely to be responsible for the accuracy of MALDI based SARS-CoV-2 diagnostics. Further analysis with more samples will be needed to validate our findings, but the methodology proves to be promising.

The protein TRIM5α has multiple roles in antiretroviral defense, but the mechanisms underlying TRIM5α action are unclear. Here, we employ APEX2-based proteomics to identify TRIM5α-interacting partners. Our proteomics results connect TRIM5 to other proteins with actions in antiviral defense. Additionally, they link TRIM5 to mitophagy, an autophagy-based mode of mitochondrial quality control that is compromised in several human diseases. We find that TRIM5 is required for Parkin-dependent and -independent mitophagy pathways where TRIM5 recruits upstream autophagy regulators to damaged mitochondria. Expression of a TRIM5 mutant lacking ubiquitin ligase activity is unable to rescue mitophagy in TRIM5 knockout cells. Cells lacking TRIM5 show reduced mitochondrial function under basal conditions and are more susceptible to immune activation and death in response to mitochondrial damage than are wild-type cells. Taken together, our studies identify a homeostatic role for a protein previously recognized exclusively for its antiviral actions.

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAPTg;Gfap+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAPTg;Gfap+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAPTg;Gfap+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAPTg;Gfap+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAPTg;Gfap+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap+ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAPTg;Gfap+/R236H mice. Last, to determine whether the findings in GFAPTg;Gfap+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAPTg;Gfap+/R236H mice.

The 2019 novel coronavirus infectious disease (COVID-19) pandemic has resulted in an unsustainable need for diagnostic tests. Currently, molecular tests are the accepted standard for the detection of SARS-CoV-2. Mass spectrometry (MS) enhanced by machine learning (ML) has recently been postulated to serve as a rapid, high-throughput, and low-cost alternative to molecular methods. Automated ML is a novel approach that could move mass spectrometry techniques beyond the confines of traditional laboratory settings. However, it remains unknown how different automated ML platforms perform for COVID-19 MS analysis. To this end, the goal of our study is to compare algorithms produced by two commercial automated ML platforms (Platforms A and B). Our study consisted of MS data derived from 361 subjects with molecular confirmation of COVID-19 status including SARS-CoV-2 variants. The top optimized ML model with respect to positive percent agreement (PPA) within Platforms A and B exhibited an accuracy of 94.9%, PPA of 100%, negative percent agreement (NPA) of 93%, and an accuracy of 91.8%, PPA of 100%, and NPA of 89%, respectively. These results illustrate the MS method's robustness against SARS-CoV-2 variants and highlight similarities and differences in automated ML platforms in producing optimal predictive algorithms for a given dataset.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual's genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.

Individuals with coronavirus disease 2019 (COVID-19) frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single-cell RNA sequencing (scRNA-seq) and cytokine analyses of cerebrospinal fluid (CSF) and blood from individuals with COVID-19 with neurological symptoms, we find compartmentalized, CNS-specific T cell activation and B cell responses. All affected individuals had CSF anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are present only during brain infection and not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from an individual with COVID-19 and found that these monoclonal antibodies (mAbs) target antiviral and antineural antigens, including one mAb that reacted to spike protein and neural tissue. CSF immunoglobulin G (IgG) from 5 of 7 patients showed antineural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of individuals with COVID-19 and suggests a role of autoimmunity in neurologic sequelae of COVID-19.

The 2019 novel coronavirus infectious disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created an unsustainable need for molecular diagnostic testing. Molecular approaches such as reverse transcription (RT) polymerase chain reaction (PCR) offers highly sensitive and specific means to detect SARS-CoV-2 RNA, however, despite it being the accepted "gold standard", molecular platforms often require a tradeoff between speed versus throughput. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) has been proposed as a potential solution for COVID-19 testing and finding a balance between analytical performance, speed, and throughput, without relying on impacted supply chains. Combined with machine learning (ML), this MALDI-TOF-MS approach could overcome logistical barriers encountered by current testing paradigms. We evaluated the analytical performance of an ML-enhanced MALDI-TOF-MS method for screening COVID-19. Residual nasal swab samples from adult volunteers were used for testing and compared against RT-PCR. Two optimized ML models were identified, exhibiting accuracy of 98.3%, positive percent agreement (PPA) of 100%, negative percent agreement (NPA) of 96%, and accuracy of 96.6%, PPA of 98.5%, and NPA of 94% respectively. Machine learning enhanced MALDI-TOF-MS for COVID-19 testing exhibited performance comparable to existing commercial SARS-CoV-2 tests.

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

Walnuts (Juglans regia L.) are a valuable dietary source of polyphenols and lipids, with increasing worldwide consumption. California is a major producer, with 'Chandler' and 'Tulare' among the cultivars more widely grown. 'Chandler' produces kernels with extra light color at a higher frequency than other cultivars, gaining preference by growers and consumers. Here we performed a deep comparative proteome analysis of kernel pellicle tissue from these two valued genotypes at three harvest maturities, detecting a total of 4937 J. regia proteins. Late and early maturity stages were compared for each cultivar, revealing many developmental responses common or specific for each cultivar. Top protein biomarkers for each developmental stage were also selected based on larger fold-change differences and lower variance among replicates, including proteins for biosynthesis of lipids and phenols, defense-related proteins and desiccation stress-related proteins. Comparison between the genotypes also revealed the common and specific protein repertoires, totaling 321 pellicle proteins with differential abundance at harvest stage. The proteomics data provides clues on antioxidant, secondary, and hormonal metabolism that could be involved in the loss of quality in the pellicles during processing for commercialization.

The interaction between the plant host, walnut (Juglans regia; Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

Plant secretome studies highlight the importance of vascular plant defense proteins against pathogens. Studies on Pierce's disease of grapevines caused by the xylem-limited bacterium Xylella fastidiosa (Xf) have detected proteins and pathways associated with its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to methodological challenges. Prosit, a deep learning neural network prediction method is a powerful tool for improving proteome profiling by data-independent acquisition (DIA). We explored the potential of Prosit's in silico spectral library predictions to improve DIA proteomic analysis of vascular leaf sap from grapevines with Pierce's disease. The combination of DIA and Prosit-predicted libraries increased the total number of identified grapevine proteins from 145 to 360 and Xf proteins from 18 to 90 compared to gas-phase fractionation (GPF) libraries. The new proteins increased the range of molecular weights, assisted in the identification of more exclusive peptides per protein, and increased identification of low-abundance proteins. These improvements allowed identification of new functional pathways associated with cellular responses to oxidative stress, to be investigated further.

Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing β-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau.AbbreviationsAMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16.

Sex estimation of skeletons is fundamental to many archaeological studies. Currently, three approaches are available to estimate sex-osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex estimation was possible in 100% of this burial sample using proteomics, in 91% using genomics, and in 51% using osteology. Agreement between the methods was high, however conflicts did occur. Genomic sex estimates were 100% consistent with proteomic and osteological estimates when DNA reads were above 100,000 total sequences. However, more than half the samples had DNA read numbers below this threshold, producing high rates of conflict with osteological and proteomic data where nine out of twenty conditional DNA sex estimates conflicted with proteomics. While the DNA signal decreased by an order of magnitude in the older burial samples, there was no decrease in proteomic signal. We conclude that proteomics provides an important complement to osteological and shotgun-genomic sex estimation.

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 μg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ± 5 to 73 ± 5 (p = 1 × 10-13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20's, 40's and 60's. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 108. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.

NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are incompletely understood. Yeast (Saccharomyces cerevisiae) cells lacking the N-terminal (Nt) protein acetyltransferase complex NatB exhibit an approximate 50% reduction in NAD+ levels and aberrant metabolism of NAD+ precursors, changes that are associated with a decrease in nicotinamide mononucleotide adenylyltransferase (Nmnat) protein levels. Here, we show that this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins and not of other NatB substrates. Nt-acetylation critically regulates protein degradation by the N-end rule pathways, suggesting that the absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t½) of non-Nt-acetylated Nmnats did not significantly differ from those of Nt-acetylated Nmnats. Accordingly, deletion or depletion of the N-end rule pathway ubiquitin E3 ligases in NatB mutants did not restore NAD+ levels. Next, we examined whether the status of Nt-acetylation would affect the translation of Nmnats, finding that the absence of Nt-acetylation does not significantly alter the polysome formation rate on Nmnat mRNAs. However, we observed that NatB mutants have significantly reduced Nmnat protein maturation. Our findings indicate that the reduced Nmnat levels in NatB mutants are mainly due to inefficient protein maturation. Nmnat activities are essential for all NAD+ biosynthesis routes, and understanding the regulation of Nmnat protein homeostasis may improve our understanding of the molecular basis and regulation of NAD+ metabolism.

Ibuprofen, an inhibitor of prostanoid biosynthesis, is a common pharmacological agent used for the management of pain, inflammation and fever. However, the chronic use of ibuprofen at high doses is associated with increased risk for cardiovascular, renal, gastrointestinal and liver injuries. The underlying mechanisms of ibuprofen-mediated effects on liver remain unclear. To determine the mechanisms and signaling pathways affected by ibuprofen (100 mg/kg/day for seven days), we performed proteomic profiling of male mice liver with quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. More than 300 proteins were significantly altered between the control and ibuprofen-treated groups. The data suggests that several major pathways including (1) energy metabolism, (2) protein degradation, (3) fatty acid metabolism and (4) antioxidant system are altered in livers from ibuprofen treated mice. Independent validation of protein changes in energy metabolism and the antioxidant system was carried out by Western blotting and showed sex-related differences. Proteasome and immunoproteasome activity/expression assays showed ibuprofen induced gender-specific proteasome and immunoproteasome dysfunction in liver. The study observed multifactorial gender-specific ibuprofen-mediated effects on mice liver and suggests that males and females are affected differently by ibuprofen.

Endomembrane damage elicits homeostatic responses including ESCRT-dependent membrane repair and autophagic removal of damaged organelles. Previous studies have suggested that these systems may act separately. Here, we show that galectin-3 (Gal3), a β-galactoside-binding cytosolic lectin, unifies and coordinates ESCRT and autophagy responses to lysosomal damage. Gal3 and its capacity to recognize damage-exposed glycans were required for efficient recruitment of the ESCRT component ALIX during lysosomal damage. Both Gal3 and ALIX were required for restoration of lysosomal function. Gal3 promoted interactions between ALIX and the downstream ESCRT-III effector CHMP4 during lysosomal repair. At later time points following lysosomal injury, Gal3 controlled autophagic responses. When this failed, as in Gal3 knockout cells, lysosomal replacement program took over through TFEB. Manifestations of this staged response, which includes membrane repair, removal, and replacement, were detected in model systems of lysosomal damage inflicted by proteopathic tau and during phagosome parasitism by Mycobacterium tuberculosis.

Plant secretome studies highlight the importance of vascular plant defense proteins against pathogens. Studies on Pierce's disease of grapevines caused by the xylem-limited bacterium Xylella fastidiosa (Xf) have detected proteins and pathways associated with its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to methodological challenges. Prosit, a deep learning neural network prediction method is a powerful tool for improving proteome profiling by data-independent acquisition (DIA). We explored the potential of Prosit's in silico spectral library predictions to improve DIA proteomic analysis of vascular leaf sap from grapevines with Pierce's disease. The combination of DIA and Prosit-predicted libraries increased the total number of identified grapevine proteins from 145 to 360 and Xf proteins from 18 to 90 compared to gas-phase fractionation (GPF) libraries. The new proteins increased the range of molecular weights, assisted in the identification of more exclusive peptides per protein, and increased identification of low-abundance proteins. These improvements allowed identification of new functional pathways associated with cellular responses to oxidative stress, to be investigated further.

The interaction between the plant host, walnut (Juglans regia; Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

Estrogen-receptor negative (ERneg) breast cancer is an aggressive breast cancer subtype in the need for new therapeutic options. We have analyzed metabolomics, proteomics and transcriptomics data for a cohort of 276 breast tumors (MetaCancer study) and nine public transcriptomics datasets using univariate statistics, meta-analysis, Reactome pathway analysis, biochemical network mapping and text mining of metabolic genes. In the MetaCancer cohort, a total of 29% metabolites, 21% proteins and 33% transcripts were significantly different (raw p

Numerous genetic conditions give rise to a scaly skin phenotype as a result of impaired barrier function. Present work investigates the degree to which the departure from normal of ichthyosis corneocytes on the skin surface depends upon the basic defect as judged by proteomic profiling. Analyzing autosomal recessive congenital ichthyosis arising from defects in the genes PNPLA1, SDR9C7 and TGM1 revealed that profiles of PNPLA1 samples displayed the greatest degree of departure from normal control epidermis, with SDR9C7 samples nearly as divergent, and TGM1 the least divergent. Although the profiles were distinctive, each displaying a set of altered protein levels, they exhibited alterations in 20 proteins in common, of which 15 were expressed consistently at higher and 5 at lower levels. Departure from the normal profile was examined at three different anatomic sites (forearm, forehead, leg). Reflecting that the normal protein profile differed at these sites, comparing profiles from afflicted subjects revealed that the degree of alteration in profile was site-dependent. These results suggest proteomic profiling can provide a quantitative measure of departure from the normal state of epidermis. Further development may help characterize consequences of the genetic defects, including perturbation of signaling pathways, and supplement visual evaluation of treatment. SIGNIFICANCE: ARCI are rare cornification disorders caused by mutations in at least 14 different genes leading to perturbed metabolism and organization of constituent biomolecules of cornified envelopes. The phenotypic manifestations of the disorder vary among individuals with the same as well as different genetic defects and even at different anatomic sites within the same individual. The present study investigates the proteomic disturbances at three anatomic sites in patients carrying mutations in three different genes. Our findings provide a basis for elucidating genotype to proteome relationships for ARCI, further investigation of which may help to delineate the underlying pathways as well as to identify new drug targets.

Trophectoderm of blastocysts mediate early events in fetal-maternal communication, enabling implantation and establishment of a functional placenta. Inadequate or impaired developmental events linked to trophoblasts directly impact early embryo survival and successful implantation during a crucial period that corresponds with high incidence of pregnancy losses in dairy cows. As yet, the molecular basis of bovine trophectoderm development and signaling towards initiation of implantation remains poorly understood. In this study, we developed methods for culturing undifferentiated bovine blastocyst-derived trophoblasts and used both transcriptomics and proteomics in early colonies to categorize and elucidate their functional characteristics. A total of 9270 transcripts and 1418 proteins were identified and analyzed based on absolute abundance. We profiled an extensive list of growth factors, cytokines and other relevant factors that can effectively influence paracrine communication in the uterine microenvironment. Functional categorization and analysis revealed novel information on structural organization, extracellular matrix composition, cell junction and adhesion components, transcription networks, and metabolic preferences. Our data showcase the fundamental physiology of bovine trophectoderm and indicate hallmarks of the self-renewing undifferentiated state akin to trophoblast stem cells described in other species. Functional features uncovered are essential for understanding early events in bovine pregnancy towards initiation of implantation.

Defects in keratinocyte transglutaminase (TGM1), resulting in an improper protein scaffold for deposition of the lipid barrier, comprise a major source of autosomal recessive congenital ichthyosis. For that reason, the composition and formation of the cornified (cross-linked) protein envelope of the epidermis have been of considerable interest. Since the isopeptide cross-linked protein components are not individually isolable once incorporated, purified envelopes were analysed by mass spectrometry after trypsin digestion. Quantitative estimates of the identified components revealed some 170 proteins, each comprising at least 0.001% of the total, of which keratins were major constituents accounting for ≈74% of the total. Some prevalent non-keratin constituents such as keratinocyte proline-rich protein, loricrin and late envelope protein-7 were preferentially incorporated into envelopes. The results suggest a model where, as previously observed in hair shaft and nail plate, a diversity of cellular proteins are incorporated. They also help rationalize the minimal effect on epidermis of ablating genes for specific single envelope structural components. The quantitative profile of constituent proteins provides a foundation for future exploration of envelope perturbations that may occur in pathological conditions.

Epidermal keratinocytes undergo cornification to form the cellular building blocks of hard skin appendages such as nails and the protective layer on the surface of the skin. Cornification requires the cross-linking of structural proteins and the removal of other cellular components to form mechanically rigid and inert corneocytes. Autophagy has been proposed to contribute to this intracellular remodelling process, but its molecular targets in keratinocytes, if any, have remained elusive. Here, we deleted the essential autophagy factor Atg7 in K14-positive epithelia of mice and determined by proteomics the impact of this deletion on the abundance of individual proteins in cornified nails. The genetic suppression of autophagy in keratinocytes resulted in a significant increase in the number of proteins that survived cornification and in alterations of their abundance in the nail proteome. A broad range of enzymes and other non-structural proteins were elevated whereas the amounts of cytoskeletal proteins of the keratin and keratin-associated protein families, cytolinker proteins and desmosomal proteins were either unaltered or decreased in nails of mice lacking epithelial autophagy. Among the various types of non-cytoskeletal proteins, the subunits of the proteasome and of the TRiC/CCT chaperonin were most strongly elevated in mutant nails, indicating a particularly important role of autophagy in removing these large protein complexes during normal cornification. Taken together, the results of this study suggest that autophagy is active during nail keratinocyte cornification and its substrate specificity depends on the accessibility of proteins outside of the cytoskeleton and their presence in large complexes.

The Ser/Thr protein kinase MTOR (mechanistic target of rapamycin kinase) regulates cellular metabolism and controls macroautophagy/autophagy. Autophagy has both metabolic and quality control functions, including recycling nutrients at times of starvation and removing dysfunctional intracellular organelles. Lysosomal damage is one of the strongest inducers of autophagy, and yet mechanisms of its activation in response to lysosomal membrane damage are not fully understood. Our recent study has uncovered a new signal transduction system based on cytosolic galectins that elicits autophagy by controlling master regulators of metabolism and autophagy, MTOR and AMPK, in response to lysosomal damage. Thus, intracellular galectins are not, as previously thought, passive tags recognizing damage to guide selective autophagy receptors, but control the activation state of AMPK and MTOR in response to endomembrane damage. Abbreviations: MTOR: mechanistic target of rapamycin kinase; AMPK: AMP-activated protein kinase / Protein Kinase AMP-Activated; SLC38A9: Solute Carrier Family 38 Member 9; APEX2: engineered ascorbate peroxidase 2; RRAGA/B: Ras Related GTP Binding A or B; LAMTOR1: Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 1; LGALS8: Lectin, Galactoside-Binding, Soluble, 8 / Galectin 8; LGALS9: Lectin, Galactoside-Binding, Soluble, 9 / Galectin 9; TAK1: TGF-Beta Activated Kinase 1 / Mitogen-Activated Protein Kinase Kinase Kinase 7 (MAP3K7); STK11/LKB1: Serine/Threonine Kinase 11 / Liver Kinase B1; ULK1: Unc-51 Like Autophagy Activating Kinase 1.

Amelogenin genes are located on both X and Y sex chromosomes in humans and are a major focus of DNA-based sex estimation methods. Amelogenin proteins, AMELX_HUMAN and AMELY_HUMAN, are expressed in the tooth organ and play a major role in mineralization of enamel, the most taphonomically resistant, archaeologically persistent human tissue. We describe shotgun liquid chromatography mass spectrometry analysis of 40 enamel samples representing 25 individuals, including modern third molars and archaeological teeth from open-air contexts including permanent adult (400 to 7300 BP) and deciduous teeth (100 to 1000 BP). Peptides specific to the X-chromosome isoform of amelogenin were detected in all samples. Peptides specific to the sexually dimorphic Y-chromosome isoform were also detected in 26 samples from 13 individuals, across all time periods, including previously unsexed deciduous teeth from archaeological contexts. While the signal of each gene product can vary by more than an order of magnitude, we show close agreement between osteological and amelogenin-based sex estimation and thus demonstrate that the protein-based signal can be obtained reliably from open-air archaeological contexts dating to at least 7300 years ago. While samples with AMELY_HUMAN peptides are unambiguously male, samples with no AMELY_HUMAN signal may either be low signal male false negative samples or female samples. In order to estimate sex in these samples we developed a probability curve of female sex as a function of the logarithm of AMELX_HUMAN signal (p < 0.0001) using logistic regression. This is also the first demonstration using proteomics to estimate sex in deciduous teeth and pushes back the application of the method to teeth that are at least 7300 years old.