Ion-current-based Proteomic Profiling of the Retina in a Rat Model of Smith-Lemli-Opitz Syndrome

Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a well-controlled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age- and sex-matched control rats (n = 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ∼40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ion-current data (e.g. signal-to-noise ratio ≥ 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS.Smith-Lemli-Opitz syndrome (SLOS)¹ is an autosomal recessive disorder associated with subnormal growth and failure to thrive, mental retardation and neurosensory deficits, and multiple congenital anomalies, including dysmorphologies (1, 2). Early epidemiological studies estimated the incidence of SLOS as 1 in 20,000 to 1 in 60,000 live births, primarily among Caucasians (1, 2). However, more recent studies suggest that the SLOS carrier frequency is ∼1 in 30 to 1 in 50; this predicts a much higher actual disease frequency, ranging from 1 in 1,590 to 1 in 17,000 (3, 4), making SLOS the fourth most common autosomal recessive human disease (after cystic fibrosis, phenylketonuria, and hemochromatosis). Mutation of the DHCR7 gene is the intrinsic cause of SLOS; this gene encodes the enzyme DHCR7 (3β-hydroxysterol-Δ7-reductase, a.k.a. 7-dehydrocholesterol reductase; EC1.3.1.21), which catalyzes the final step in the cholesterol biosynthetic pathway, reducing the Δ7 double bond and thus converting 7-dehydrocholesterol (7DHC) to cholesterol (4, 5). As a consequence, markedly reduced levels of cholesterol and aberrantly elevated levels of the cholesterol precursor 7DHC (and its epimer, 8DHC) are observed in the majority of affected SLOS patients (6, 7). Therefore, the clinical suspicion of SLOS is confirmed by elevated 7DHC in plasma or tissues, typically demonstrated via chromatographic methods (e.g. HPLC or GC/MS) (8, 9).Visual capacity may become compromised in SLOS patients because of a variety of congenital or postnatal pathologies, such as cataracts, aniridia, corneal endothelium defects, sclerocornea, electrophysiological defects in the retina, optic nerve abnormalities, or other ophthalmologic problems (10, 11). We currently lack full knowledge of the exact pathobiological mechanism underlying SLOS, but additional insights may be afforded by studies employing a rodent model of the disease in which rats are treated with AY9944 (trans-1,4-bis[2-chlorobenzylaminomethyl] cyclohexane dihydrochloride), a relatively selective inhibitor of DHCR7 (12–14). We previously described progressive retinal degeneration in this rat model of SLOS, which is characterized by the shortening of retinal rod outer segments, pyknosis and thinning of the outer nuclear layer (ONL) of the retina (which contains the photoreceptor nuclei), and accumulation of membranous/lipid inclusions in the retinal pigment epithelium (RPE) (12, 13). Reduced rod outer segment membrane fluidity, primarily caused by a dramatic (30 to 40 mol%) decline in docosahexaenoic acid (22:6, n3) levels relative to age-matched controls, also was observed in the SLOS rat model by three postnatal months (15, 16). Retinal function and sterol steady-state in the same rat model of SLOS can be partially rescued using a high-cholesterol diet (2% by weight), although histological degeneration of the retina still occurs (17). However, the molecular mechanisms that underlie the observed electrophysiological defects in the retina, the accumulation of membranous/lipid inclusions in the RPE, the shortening of retinal rod outer segments, and the initiation of ONL pyknosis in the SLOS rat model remain poorly understood. Therefore, a comprehensive profiling of the retinal proteomes of AY9944-treated versus age-matched untreated control rats may contribute to further understanding of the underlying mechanisms responsible for the retinopathy associated with the SLOS model and, by extension, the human disease.Nevertheless, extensive and reliable expression profiling of the retinal proteome remains a prominent challenge, owing to the need to quantify data from multiple animals and a high percentage of integral membrane and membrane-associated proteins (18, 19). Label-free approaches can compare multiple replicates (20–22) with quantitative accuracy comparable to that attained with stable isotope-labeling methods (23–25). However, in order to achieve reliable relative quantification, highly quantitative and reproducible sample preparation and LC/MS analysis are required for relatively large-scale sample cohorts.In the present study, we performed a reproducible, well-controlled, ion-current-based comparative proteomic analysis of the retinas from AY9944-treated versus age/sex-matched control rats (n = 5 animals per group). A high-concentration detergent mixture was used for the efficient extraction of proteins from retinas, and samples then underwent a reproducible precipitation/on-pellet-digestion procedure and long-column, 7-h nano-LC-MS analysis. These approaches ensured extensive comparative analysis of retina samples with 10 animals. The preparative and analytical procedures were carefully optimized and controlled to ensure optimal reproducibility. Two label-free approaches, the ion-current-based method and a spectral counting method, were compared in parallel. The altered proteins were subjected to functional annotation, and selected groups of proteins of interest were further validated by means of Western blot and correlative immunohistochemical analysis.     

Quantitative Proteomic Analysis Reveals Metabolic Alterations,Calcium Dysregulation,and Increased Expression of Extracellular Matrix Proteins in Laminin α2 Chain–deficient Muscle

Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain–deficient dy^3K/dy^3K mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain–deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).Congenital muscular dystrophy with laminin α2 chain deficiency, also known as MDC1A,¹ is a severe muscle wasting disease for which there is no cure. MDC1A is caused by mutations in the LAMA2 gene that lead to complete or partial deficiency of laminin α2 chain (1–3). Although the primary defect in MDC1A is known, the secondary molecular mechanisms eventually leading to muscle degeneration are not fully understood. In normal muscle, laminin α2 chain binds to the cell surface receptors dystroglycan and integrin α7β1, which both indirectly bind the cytoskeleton (4–7). Both of these adhesion complexes are important for normal skeletal muscle function, and laminin α2 chain binding to dystroglycan contributes to the maintenance of sarcolemmal integrity and protects muscles from damage (8), whereas laminin α2 chain binding to integrin α7β1 promotes myofiber survival (9, 10). In MDC1A, laminin α2 chain is absent or severely reduced, and the expression of dystroglycan and α7β1 is also dysregulated in MDC1A (9, 11, 12). Thus, the structural link is broken, and the yet to be determined downstream intracellular signaling pathways are also interrupted. Consequently, laminin α2 chain–deficient muscle fibers undergo degeneration–regeneration cycles, but rather quickly regeneration fails and muscle fibers die by apoptosis/necrosis followed by a major replacement of muscle tissue with connective tissue (3, 7). In order to unravel novel secondary molecular mechanisms, which could indicate new therapeutic targets, we decided to evaluate the protein expression profile in laminin α2 chain–deficient dy^3K/dy^3K muscle. Several proteomic profiling studies of dystrophin-deficient muscles (Duchenne muscular dystrophy) have been performed (13–20), as well as some with dysferlin-deficient muscles (Limb-girdle muscular dystrophy type 2B, Miyoshi myopathy) (21, 22). They all showed a great number of proteins that were differentially expressed in different dystrophic muscles and at different ages (13–22). However, proteomic analyses of laminin α2 chain–deficient muscle have not yet been performed. We here used multidimensional protein identification technology with tandem mass tags (TMT), a powerful shotgun label-based proteomic method that separates peptides in two-dimensional liquid chromatography (23, 24). We identified around 100 proteins that were differentially expressed in laminin α2 chain–deficient gastrocnemius and diaphragm muscles relative to the corresponding wild-type muscles, and the differential expression of selected proteins was verified with Western blot analysis or immunofluorescence.     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.

Polychlorinated biphenyls (PCBs) are produced by chlorination of biphenyl, resulting in up to 209 different congeners. Commercial mixtures range from light oily fluids to waxes, and their physical properties make them useful as heat transfer fluids, hydraulic fluids, solvent extenders, plasticizers, flame retardants, organic diluents, and dielectric fluids (1, 21). Approximately 24 million lb are in the North American environment (19). The stability and hydrophobic nature of these compounds make them a persistent environmental hazard.To date, bacterial transformations have been the main focus of PCB degradation research. Aerobic bacteria use a biphenyl-induced dioxygenase enzyme system to attack less-chlorinated congeners (mono- to hexachlorobiphenyls) (1, 5, 7, 8, 22). Although more-chlorinated congeners are recalcitrant to aerobic bacterial degradation, microorganisms in anaerobic river sediments reductively dechlorinate these compounds, mainly removing the meta and para chlorines (1, 6, 10, 33, 34).The degradation of PCBs by white rot fungi has been known since 1985 (11, 18). Many fungi have been tested for their ability to degrade PCBs, including the white rot fungi Coriolus versicolor (18), Coriolopsis polysona (41), Funalia gallica (18), Hirneola nigricans (35), Lentinus edodes (35), Phanerochaete chrysosporium (3, 11, 14, 17, 18, 35, 39, 41–43), Phlebia brevispora (18), Pleurotus ostreatus (35, 43), Poria cinerescens (18), Px strain (possibly Lentinus tigrinus) (35), and Trametes versicolor (41, 43). There have also been studies of PCB metabolism by ectomycorrhizal fungi (17) and other fungi such as Aspergillus flavus (32), Aspergillus niger (15), Aureobasidium pullulans (18), Candida boidinii (35), Candida lipolytica (35), Cunninghamella elegans (16), and Saccharomyces cerevisiae (18, 38). The mechanism of PCB biodegradation has not been definitively determined for any fungi. White rot fungi produce several nonspecific extracellular enzymes which have been the subject of extensive research. These nonspecific peroxidases are normally involved in lignin degradation but can oxidize a wide range of aromatic compounds including polycyclic aromatic hydrocarbons (37). Two peroxidases, lignin peroxidase (LiP) and Mn peroxidase (MnP), are secreted into the environment of the fungus under conditions of nitrogen limitation in P. chrysosporium (23, 25, 27, 29) but are not stress related in fungi such as Bjerkandera adusta or T. versicolor (12, 30).Two approaches have been used to determine the biodegradability of PCBs by fungi: (i) loss of the parent congener analyzed by gas chromatography (GC) (17, 32, 35, 42, 43) and (ii) mineralization experiments in which the ¹⁴C of the universally labeled ¹⁴C parent congener is recovered as ¹⁴CO₂ (11, 14, 18, 39, 41). In the first method, the loss of a peak on a chromatogram makes it difficult to decide whether the PCB is being partly degraded, mineralized, adsorbed to the fungal biomass, or bound to glassware, soil particles, or wood chips. Even when experiments with killed-cell and abiotic controls are performed, the extraction efficiency and standard error can make data difficult to interpret. For example, recoveries can range anywhere from 40 to 100% depending on the congener used and the fungus being investigated (17). On the other hand, recovery of significant amounts of ¹⁴CO₂ from the cultures incubated with a ¹⁴C substrate provides definitive proof of fungal metabolism. There appears to be only one report relating data from these two techniques (18), and in that study, [U-¹⁴C]Aroclor 1254, rather than an individual congener, was used.In this study, we examined the ability of 12 white rot fungal strains to metabolize selected PCB congeners to determine which strains were the most active degraders. Included in this group was P. chrysosporium ATCC 24725, a strain used extensively in PCB studies (3, 14, 18, 35, 39, 41–43). Six PCB congeners were selected to give a range of chlorine substitutions and therefore a range of potential biodegradability which was monitored by GC. One of the chosen congeners was ¹⁴C labeled and used in studies to compare the results from a mineralization method with those from the GC method.     

Biochemical and Genetic Characterization of a Gentisate 1,2-Dioxygenase from Sphingomonas sp. Strain RW5

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488–6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (K_m) and specificity constants (K_cat/K_m) of the enzyme were determined to be 15 μM and 511 s⁻¹ M⁻¹ × 10⁴ for gentisate and 754 μM and 20 s⁻¹ M⁻¹ × 10⁴ for 3,6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.Large amounts of aromatic compounds have been released into the environment during the last decades as a result of extensive production of industrial chemicals and agricultural applications of pesticides. Many of these compounds, particularly the chlorinated derivatives, are toxic, even at low concentrations, and persist in the environment (14, 39). Numerous microorganisms have been isolated which degrade xenobiotic aromatic compounds through aerobic or anaerobic degradative reactions (16, 17, 34, 46, 47). A wide variety of polycyclic and homocyclic aromatic compounds are aerobically transformed to a limited number of central dihydroxylated intermediates like catechol, protocatechuate, or gentisate. Whereas catabolic pathways for catechol and protocatechuate have been extensively characterized (22, 35), little is known about gentisate degradation.Gentisic acid (2,5-dihydroxybenzoic acid) is a key intermediate in the aerobic degradation of such aromatic compounds as dibenzofuran (15), naphthalene (18, 48), salicylate (40, 55), anthranilate (32), and 3-hydroxybenzoate (26). Degradation of gentisate is initiated by gentisate 1,2-dioxygenase (GDO; EC 1.13.11.4, gentisate:oxygen oxidoreductase), which cleaves the aromatic ring between the carboxyl and the vicinal hydroxyl group to form maleylpyruvate (30). Maleylpyruvate can be converted to central metabolites of the Krebs cycle either by cleavage to pyruvate and maleate (5, 24) or by isomerization to fumarylpyruvate and subsequent cleavage to fumarate and pyruvate (10, 31, 51).Of the two well-studied classes of ring cleavage dioxygenases, intradiol enzymes, such as catechol 1,2-dioxygenase or protocatechuate 3,4-dioxygenase, contain an Fe³⁺ atom in the catalytic center and cleave the aromatic substrate between two vicinal hydroxyl groups (7, 37, 38), whereas dioxygenases of the extradiol class, such as catechol 2,3-dioxygenase or protocatechuate 4,5-dioxygenase, contain Fe²⁺ and cleave the aromatic substrate adjacent to two vicinal hydroxyl groups (1, 13). Gentisate 1,2-dioxygenase cleaves aromatic rings containing hydroxyl groups situated para to one another. Although the mechanism of oxygen activation was proposed to be similar to that of enzymes of the extradiol dioxygenase class (20), and the active center contains Fe²⁺ (11, 21, 29, 49, 51), the Fe²⁺ is not bound to the enzyme by electron-donating ligands such as cysteine or tyrosine (21) as is the case for extradiol-cleaving dioxygenases (19). It is being assumed, therefore, that GDO represents a novel class of ring-cleaving dioxygenases.GDOs have been purified and characterized from gram-positive bacteria of the genera Bacillus and Rhodococcus (29, 50) and gram-negative bacteria of the genera Klebsiella, Comamonas, and Moraxella (11, 21, 49), and amino-terminal sequences of GDOs from Comamonas testosteroni and Comamonas acidovorans have been determined (21), but until now, no complete sequence of any GDO or of a gene encoding GDO has been reported. Here we describe the cloning and sequencing of the gene encoding the GDO from Sphingomonas sp. strain RW5 and its partial characterization. This GDO represents a novel class of dioxygenases with very low similarity to any other known ring-cleaving dioxygenases.     

Proteome-derived Peptide Libraries Allow Detailed Analysis of the Substrate Specificities of Nα-acetyltransferases and Point to hNaa10p as the Post-translational Actin Nα-acetyltransferase

The impact of N^α-terminal acetylation on protein stability and protein function in general recently acquired renewed and increasing attention. Although the substrate specificity profile of the conserved enzymes responsible for N^α-terminal acetylation in yeast has been well documented, the lack of higher eukaryotic models has hampered the specificity profile determination of N^α-acetyltransferases (NATs) of higher eukaryotes. The fact that several types of protein N termini are acetylated by so far unknown NATs stresses the importance of developing tools for analyzing NAT specificities. Here, we report on a method that implies the use of natural, proteome-derived modified peptide libraries, which, when used in combination with two strong cation exchange separation steps, allows for the delineation of the in vitro specificity profiles of NATs. The human NatA complex, composed of the auxiliary hNaa15p (NATH/hNat1) subunit and the catalytic hNaa10p (hArd1) and hNaa50p (hNat5) subunits, cotranslationally acetylates protein N termini initiating with Ser, Ala, Thr, Val, and Gly following the removal of the initial Met. In our studies, purified hNaa50p preferred Met-Xaa starting N termini (Xaa mainly being a hydrophobic amino acid) in agreement with previous data. Surprisingly, purified hNaa10p preferred acidic N termini, representing a group of in vivo acetylated proteins for which there are currently no NAT(s) identified. The most prominent representatives of the group of acidic N termini are γ- and β-actin. Indeed, by using an independent quantitative assay, hNaa10p strongly acetylated peptides representing the N termini of both γ- and β-actin, and only to a lesser extent, its previously characterized substrate motifs. The immunoprecipitated NatA complex also acetylated the actin N termini efficiently, though displaying a strong shift in specificity toward its known Ser-starting type of substrates. Thus, complex formation of NatA might alter the substrate specificity profile as compared with its isolated catalytic subunits, and, furthermore, NatA or hNaa10p may function as a post-translational actin N^α-acetyltransferase.The multisubunit and ribosome-associated protein N^α-acetyltransferases (NATs)1 are omnipresent enzyme complexes that catalyze the transfer of the acetyl moiety from acetyl-CoA to the primary α-amines of N termini of nascent proteins (1–3). As up to 50 to 60% of yeast proteins and 80 to 90% of human proteins are modified in this manner, N^α-acetylation is a widespread protein modification in eukaryotes (4–7), and the pattern of modification has remained largely conserved throughout evolution (4, 8). NATs belong to a subfamily of the Gcn5-related N-acetyltransferase superfamily of N-acetyltransferases, additionally encompassing the well-studied histone acetyltransferases that are implicated in epigenetic imprinting.In yeast and humans, three main NAT complexes, NatA, NatB, and NatC were found to be responsible for the majority of N^α-terminal acetylations (1). The NatA complex, responsible for cotranslational N^α-terminal acetylation of proteins with Ser, Ala, Thr, Gly, and Val N termini, is composed of two main subunits, the catalytic subunit Naa10p (previously known as Ard1p) and the auxiliary subunit Naa15p (previously known as Nat1p/NATH) (9–11). Furthermore, a third catalytic subunit Naa50p (previously known as Nat5)—an acetyltransferase shown to function in chromosome cohesion and segregation (12–14)—was found to physically interact with the NatA complex of yeast (2), fruit fly (12), and human (15). Recently, human Naa50p (hNaa50p) was reported to display lysine or N^ε-acetyltransferase as well as NAT activity (16), the latter was defined as NatE activity (16). Interestingly, the chaperone-like, Huntingtin interacting protein HYPK, identified as a novel stable interactor of human NatA, was functionally implicated in the N-terminal acetylation of an in vivo NatA substrate, demonstrating that NAT complex formation and composition may have an overall influence on the observed (degree of) N^α-acetylation (17). Further, subunits of the human NatA complex have been coupled to cancer-related processes and differentiation, with altered subunit expression reported in papillary thyroid carcinoma, neuroblastoma, and retinoic acid induced differentiation. Furthermore, the NatA catalytic subunit was found to be implicated in processes such as hypoxia-response and the β-catenin pathway (18, 19). Of note is that in line with the differential localization patterns of the individual NatA subunits (9, 13, 20, 21), other data indicate that these subunits might well exert NatA-independent enzymatic functions (13, 22, 23). Given that a significant fraction of hNaa10p and hNaa15p are nonribosomal (9), and given the multitude of postulated post-translational in vivo N-acetylation events recently reported (24–26), these observations argue in favor of the existence of NAT complexes and/or catalytic NAT-subunits acting post-translationally.Similar to NatA, the NatB and NatC complexes, composed of the catalytic subunit Naa20p or Naa30p and the auxiliary subunits Naa25p or Naa35p and Naa38p respectively, are conserved from yeast to higher eukaryotes concerning their subunit composition as well as their substrate specificity. Both these complexes display activity toward methionine-starting N termini, with NatB preferring acidic residues as well as Asn and Gln at P2′-sites², whereas NatC prefers hydrophobic amino acid residues at substrate P2′-sites (1, 27, 28).N^α-acetylation affects various protein functions such as localization, activity, association, and stability (29, 30). Only recently a more generalized function of protein N^α-acetylation in generating so-called N-terminal degrons marking proteins for removal was put forward (31). The lack of mouse models in addition to the fact that (combined) knockdown of individual components of N^α-acetyltransferases only marginally affect the overall N^α-acetylation status (4) have so far hampered the molecular characterization of the substrate specificity profile of (yet uncharacterized) NATs. To date, all eukaryote N^α-acetylation events are assumed to be catalyzed by the five known NATs (32). However, an additional level of complexity is imposed by the fact that in contrast to yeast, higher eukaryotes express multiple splice variants of various NAT subunits as well as paralogs thereof (33, 34), further implicating that a specific NAT''s substrate specificity might be altered in this way, in addition to the possible existence of substrate redundancy. Moreover, regulation of substrate specificity and stability of NAT activity can be imposed by differential complex formation and post-translational modifications including phosphorylation, auto-acetylation, and specific proteolytic cleavage of the catalytic subunits (9, 16, 17). As such, a detailed understanding of the substrate specificity of NATs, and the regulation thereof, could help unravel the physiological substrate repertoires as well as the associated physiological roles of NATs in the normal and the disease state.The specificity of N^α-acetyltransferases and their endogenous substrates were originally studied by two-dimensional-PAGE: N^α-acetylation neutralizes the N-terminal positive charge, resulting in an altered electrophoretic protein migration during isoelectric focusing (35–38). Recently, this altered biophysical property was also exploited to enrich for protein N-termini using low pH strong cation exchange (SCX) chromatography (24, 39). As an example, SCX prefractionation combined with N-terminal combined fractional diagonal chromatography, a targeted proteomics technology negatively selecting for protein N-terminal peptides, stable isotope labeling of amino acids in cell culture, and amino-directed modifiers (40), was used to study the in vivo substrate repertoires of human as well as yeast NatA (4).Nevertheless, the various methods reported today to study in detail N^α-terminal acetylation and thus the specificities of different NATs make use of a limited and therefore somewhat biased set of synthesized peptide substrates and comprise the rather laborious detection of radioactive acetylated products as well as enzyme-coupled methods quantifying acetyl-CoA conversion. Because (proteome-derived) peptide libraries have been used extensively to study epitope mapping (41), protein-protein interactions (42), protein modifications such as phosphorylation (43), and proteolysis (44, 45), as well as for determining the substrate specificity of the N^α-deblocking peptide deformylase (46), we reckoned that the development of an oligopeptide-based acetylation assay should allow for more comprehensive screening of NAT-like activities. We here report on the development of a peptide-based method to systematically screen for the in vitro sequence specificity profile of individual NATs as well as endogenous NAT complexes. In summary, SCX enriched, N^α-free peptide libraries, derived from natural proteomes build up the peptide substrate pool. And, upon incubation, NAT N^α-acetylated peptides are enriched by a second SCX fractionation step, resulting in a positive selection of NAT-specific peptide substrates. By use of this proteome-derived peptide library approach, we here delineated (differences in) the specificity profiles of hNaa50p and hNaa10p as isolated hNatA components, as well as of assayed their combined activity when in their native hNatA complex.     

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein–protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.Protein–protein interactions (PPIs)¹ play a key role in defining protein functions in biological systems. Aberrant PPIs can have drastic effects on biochemical activities essential to cell homeostasis, growth, and proliferation, and thereby lead to various human diseases (1). Consequently, PPI interfaces have been recognized as a new paradigm for drug development. Therefore, mapping PPIs and their interaction interfaces in living cells is critical not only for a comprehensive understanding of protein function and regulation, but also for describing the molecular mechanisms underlying human pathologies and identifying potential targets for better therapeutics.Several strategies exist for identifying and mapping PPIs, including yeast two-hybrid, protein microarray, and affinity purification mass spectrometry (AP-MS) (2–5). Thanks to new developments in sample preparation strategies, mass spectrometry technologies, and bioinformatics tools, AP-MS has become a powerful and preferred method for studying PPIs at the systems level (6–9). Unlike other approaches, AP-MS experiments allow the capture of protein interactions directly from their natural cellular environment, thus better retaining native protein structures and biologically relevant interactions. In addition, a broader scope of PPI networks can be obtained with greater sensitivity, accuracy, versatility, and speed. Despite the success of this very promising technique, AP-MS experiments can lead to the loss of weak/transient interactions and/or the reorganization of protein interactions during biochemical manipulation under native purification conditions. To circumvent these problems, in vivo chemical cross-linking has been successfully employed to stabilize protein interactions in native cells or tissues prior to cell lysis (10–16). The resulting covalent bonds formed between interacting partners allow affinity purification under stringent and fully denaturing conditions, consequently reducing nonspecific background while preserving stable and weak/transient interactions (12–16). Subsequent mass spectrometric analysis can reveal not only the identities of interacting proteins, but also cross-linked amino acid residues. The latter provides direct molecular evidence describing the physical contacts between and within proteins (17). This information can be used for computational modeling to establish structural topologies of proteins and protein complexes (17–22), as well as for generating experimentally derived protein interaction network topology maps (23, 24). Thus, cross-linking mass spectrometry (XL-MS) strategies represent a powerful and emergent technology that possesses unparalleled capabilities for studying PPIs.Despite their great potential, current XL-MS studies that have aimed to identify cross-linked peptides have been mostly limited to in vitro cross-linking experiments, with few successfully identifying protein interaction interfaces in living cells (24, 25). This is largely because XL-MS studies remain challenging due to the inherent difficulty in the effective MS detection and accurate identification of cross-linked peptides, as well as in unambiguous assignment of cross-linked residues. In general, cross-linked products are heterogeneous and low in abundance relative to non-cross-linked products. In addition, their MS fragmentation is too complex to be interpreted using conventional database searching tools (17, 26). It is noted that almost all of the current in vivo PPI studies utilize formaldehyde cross-linking because of its membrane permeability and fast kinetics (10–16). However, in comparison to the most commonly used amine reactive NHS ester cross-linkers, identification of formaldehyde cross-linked peptides is even more challenging because of its promiscuous nonspecific reactivity and extremely short spacer length (27). Therefore, further developments in reagents and methods are urgently needed to enable simple MS detection and effective identification of in vivo cross-linked products, and thus allow the mapping of authentic protein contact sites as established in cells, especially for protein complexes.Various efforts have been made to address the limitations of XL-MS studies, resulting in new developments in bioinformatics tools for improved data interpretation (28–32) and new designs of cross-linking reagents for enhanced MS analysis of cross-linked peptides (24, 33–39). Among these approaches, the development of new cross-linking reagents holds great promise for mapping PPIs on the systems level. One class of cross-linking reagents containing an enrichment handle have been shown to allow selective isolation of cross-linked products from complex mixtures, boosting their detectability by MS (33–35, 40–42). A second class of cross-linkers containing MS-cleavable bonds have proven to be effective in facilitating the unambiguous identification of cross-linked peptides (36–39, 43, 44), as the resulting cross-linked products can be identified based on their characteristic and simplified fragmentation behavior during MS analysis. Therefore, an ideal cross-linking reagent would possess the combined features of both classes of cross-linkers. To advance the study of in vivo PPIs, we have developed a new XL-MS platform based on a novel membrane-permeable, enrichable, and MS-cleavable cross-linker, Azide-A-DSBSO (azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide), and multistage tandem mass spectrometry (MSⁿ). This new XL-MS strategy has been successfully employed to map in vivo PPIs from mammalian cells at both the proteome scale and the targeted protein complex level.     

Localization and Targeting of an Unusual Pyridine Nucleotide Transhydrogenase in Entamoeba histolytica

Pyridine nucleotide transhydrogenase (PNT) catalyzes the direct transfer of a hydride-ion equivalent between NAD(H) and NADP(H) in bacteria and the mitochondria of eukaryotes. PNT was previously postulated to be localized to the highly divergent mitochondrion-related organelle, the mitosome, in the anaerobic/microaerophilic protozoan parasite Entamoeba histolytica based on the potential mitochondrion-targeting signal. However, our previous proteomic study of isolated phagosomes suggested that PNT is localized to organelles other than mitosomes. An immunofluorescence assay using anti-E. histolytica PNT (EhPNT) antibody raised against the NADH-binding domain showed a distribution to the membrane of numerous vesicles/vacuoles, including lysosomes and phagosomes. The domain(s) required for the trafficking of PNT to vesicles/vacuoles was examined by using amoeba transformants expressing a series of carboxyl-terminally truncated PNTs fused with green fluorescent protein or a hemagglutinin tag. All truncated PNTs failed to reach vesicles/vacuoles and were retained in the endoplasmic reticulum. These data indicate that the putative targeting signal is not sufficient for the trafficking of PNT to the vesicular/vacuolar compartments and that full-length PNT is necessary for correct transport. PNT displayed a smear of >120 kDa on SDS-PAGE gels. PNGase F and tunicamycin treatment, chemical degradation of carbohydrates, and heat treatment of PNT suggested that the apparent aberrant mobility of PNT is likely attributable to its hydrophobic nature. PNT that is compartmentalized to the acidic compartments is unprecedented in eukaryotes and may possess a unique physiological role in E. histolytica.Pyridine nucleotide transhydrogenase (PNT) participates in the bioenergetic processes of the cell. PNT generally resides on the cytoplasmic membranes of bacteria and the inner membrane of mammalian mitochondria (3, 16) and utilizes the electrochemical proton gradient across the membrane to drive NADPH formation from NADH (14, 15, 39) according to the reaction H⁺out + NADH + NADP⁺↔H⁺in + NAD⁺ + NADPH, where “out” and “in” denote the cytosol and the matrix of the mitochondria, or the periplasmic space and the cytosol of bacteria, respectively.PNT has been identified in several protozoan parasites, including Entamoeba histolytica (8, 51), Eimeria tenella (17, 47), Mastigamoeba balamuthi (11) Plasmodium falciparum (10), Plasmodium yoelii (6), and Plasmodium berghei (12). In general, PNT contains conserved structural units consisting of three domains, the NAD(H)-binding domain (domain I [dI]) and the NADP(H)-binding domain (domain III [dIII]), both of which face the matrix side of the eukaryotic mitochondria or the cytoplasmic side in bacteria, and the hydrophobic domain (domain II [dII]), containing 11 to 13 transmembrane regions. PNT from E. tenella and E. histolytica exists as a single polypeptide in an unusual configuration consisting of dIIb-dIII-dI-dIIa, with a 38-amino-acid-long linker region between dIII and dI (48).E. histolytica, previously considered an “amitochondriate” protist, is currently considered to possess a mitochondrion-related organelle with reduced and divergent functions, the mitosome (1, 21, 23a, 26, 42). Our recent proteomic study of isolated mitosomes identified about 20 new constituents (26), together with four proteins previously demonstrated in E. histolytica mitosomes: Cpn60 (8, 19, 21, 42), Cpn10 (46), mitochondrial Hsp70 (2, 44), and mitochondrion carrier family (MCF) (ADP/ATP transporter) (7). Despite the early presumption of PNT being localized in mitosomes (8), based on the amino-terminal region rich in hydroxylated (five serines and threonines) and acidic (three glutamates) amino acids, which slightly resembles known mitochondrion- and hydrogenosome-targeting sequences (8, 35), PNT was not discovered in the mitosome proteome. We also doubted this premise because PNT was one of the major proteins identified in isolated phagosomes (32, 33). Thus, the intracellular localization and trafficking of PNT remain unknown.In this report, we showed that E. histolytica PNT (EhPNT) is localized to various vesicles and vacuoles, including lysosomes and phagosomes, using wild-type amoebae and antiserum raised against recombinant EhPNT and an E. histolytica line expressing EhPNT with a carboxyl-terminal hemagglutinin (HA) epitope tag and anti-HA antibody. We also showed that all domains of EhPNT are required for its trafficking to the acidic compartment by using amoeba transformants expressing the HA tag or green fluorescent protein (GFP) fused with a region containing various domains of EhPNT.