Changes In Post-translational Processing Research Articles

Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Most human proteins are post-translationally modified from simple N-terminal acetylation to complex phosphorylation, lipidation and glycosylation patterns. These processes, among others, result in multiple natural proteoforms of each protein. Conversely, aberrant protein modifications can result from changes in post-translational processing and protein catabolism, xenobiotics and genetic mutation. Changes in protein amino acid composition can lead to changes in their secondary, tertiary and quaternary structures, including aggregation. In the first part of this perspective article, Jeffery Agar and co-worker demonstrate that diverse Cu/Zn-superoxide dismutase protein mutations result in a common structural consequence that can lead to toxic protein aggregation. In the second part of this perspective, they present evidence of both toxic and protective post-translational modifications in human tissue, and how these informed our current therapeutic strategies. Dr. Jeffrey Agar is Associate Professor of Chemistry at Northeastern University (Boston, MA). His research interests are centered on the characterization of proteins post-translational modifications and changes in protein, peptide, and lipid expression that occur during in disease, more specifically amyotrophic lateral sclerosis.

Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis

Enoxacin has been identified as a small molecule inhibitor of binding between the B2-subunit of vacuolar H+-ATPase (V-ATPase) and microfilaments. It inhibits bone resorption by calcitriol-stimulated mouse marrow cultures. We hypothesized that enoxacin acts directly and specifically on osteoclasts by disrupting the interaction between plasma membrane-directed V-ATPases, which contain the osteoclast-selective a3-subunit of V-ATPase, and microfilaments. Consistent with this hypothesis, enoxacin dose-dependently reduced the number of multinuclear cells expressing tartrate-resistant acid phosphatase (TRAP) activity produced by RANK-L-stimulated osteoclast precursors. Enoxacin (50 μM) did not induce apoptosis as measured by TUNEL and caspase-3 assays. V-ATPases containing the a3-subunit, but not the "housekeeping" a1-subunit, were isolated bound to actin. Treatment with enoxacin reduced the association of V-ATPase subunits with the detergent-insoluble cytoskeleton. Quantitative PCR revealed that enoxacin triggered significant reductions in several osteoclast-selective mRNAs, but levels of various osteoclast proteins were not reduced, as determined by quantitative immunoblots, even when their mRNA levels were reduced. Immunoblots demonstrated that proteolytic processing of TRAP5b and the cytoskeletal protein L-plastin was altered in cells treated with 50 μM enoxacin. Flow cytometry revealed that enoxacin treatment favored the expression of high levels of DC-STAMP on the surface of osteoclasts. Our data show that enoxacin directly inhibits osteoclast formation without affecting cell viability by a novel mechanism that involves changes in posttranslational processing and trafficking of several proteins with known roles in osteoclast function. We propose that these effects are downstream to blocking the binding interaction between a3-containing V-ATPases and microfilaments.

Different Requirements for Proteolytic Processing of Bone Morphogenetic Protein 5/6/7/8 Ligands in Drosophila melanogaster

Bone morphogenetic proteins (BMPs) are synthesized as proproteins that undergo proteolytic processing by furin/subtilisin proprotein convertases to release the active ligand. Here we study processing of BMP5/6/7/8 proteins, including the Drosophila orthologs Glass Bottom Boat (Gbb) and Screw (Scw) and human BMP7. Gbb and Scw have three functional furin/subtilisin proprotein convertase cleavage sites; two between the prodomain and ligand domain, which we call the Main and Shadow sites, and one within the prodomain, which we call the Pro site. In Gbb each site can be cleaved independently, although efficient cleavage at the Shadow site requires cleavage at the Main site, and remarkably, none of the sites is essential for Gbb function. Rather, Gbb must be processed at either the Pro or Main site to produce a functional ligand. Like Gbb, the Pro and Main sites in Scw can be cleaved independently, but cleavage at the Shadow site is dependent on cleavage at the Main site. However, both Pro and Main sites are essential for Scw function. Thus, Gbb and Scw have different processing requirements. The BMP7 ligand rescues gbb mutants in Drosophila, but full-length BMP7 cannot, showing that functional differences in the prodomain limit the BMP7 activity in flies. Furthermore, unlike Gbb, cleavage-resistant BMP7, although non-functional in rescue assays, activates the downstream signaling cascade and thus retains some functionality. Our data show that cleavage requirements evolve rapidly, supporting the notion that changes in post-translational processing are used to create functional diversity between BMPs within and between species.

In Vitro Molecular Structure of N-Terminal B-Type Natriuretic Peptide: Monomer or Oligomer?

To the Editor: In the circulation, the A-type natriuretic peptide and the B-type natriuretic peptide (BNP) regulate cardiovascular homeostasis. Each hormone is derived from biosynthetic precursors that are processed to the active C-terminal hormones and the N-terminal fragments (1). Measurements of both BNP and N-terminal proBNP in plasma are recommended as diagnostic tools for heart failure. The peptides are measured by immunoassays that use antibodies directed against specific epitopes. A basic understanding of the native peptide structure in terms of posttranslational modifications is thus paramount, because changes in posttranslational processing will affect assay measurement and clinical interpretation drastically. A recent report in Clinical Chemistry addressed the possibility of natriuretic peptide oligomerization in a study that used a recombinant N-terminal proBNP peptide (possessing a glycine–serine N-terminal extension) spiked into 3 matrices (2). Buffer, plasma, or serum was incubated for various times and temperatures; the material was then applied to a reversed-phase column, a gel-filtration column, and a reversed-phase column, respectively. All 3 columns were eluted with …

SNPeffect v2.0: a new step in investigating the molecular phenotypic effects of human non-synonymous SNPs

Single nucleotide polymorphisms (SNPs) constitute the most fundamental type of genetic variation in human populations. About 75 000 of these reported variations cause an amino acid change in the translated protein. An important goal in genomic research is to understand how this variability affects protein function, and whether or not particular SNPs are associated to disease susceptibility. Accordingly, the SNPeffect database uses sequence- and structure-based bioinformatics tools to predict the effect of non-synonymous SNPs on the molecular phenotype of proteins. SNPeffect analyses the effect of SNPs on three categories of functional properties: (1) structural and thermodynamic properties affecting protein dynamics and stability (2) the integrity of functional and binding sites and (3) changes in posttranslational processing and cellular localization of proteins. The search interface of the database can be used to search specifically for polymorphisms that are predicted to cause a change in one of these properties. Now based on the Ensembl human databases, the SNPeffect database has been remodeled to better fit an automatically updatable structure. The current edition holds the molecular phenotype of 74 567 nsSNPs in 23 426 proteins. SNPeffect can be accessed through http://snpeffect.vib.be.

Pb 2+ exposure alters the lens αA-crystallin protein profile in vivo and induces cataract formation in lens organ culture

Epidemiological data supports lead exposure as a risk factor for cataract development. Previous studies which demonstrated oxidative imbalances in the lens following in vivo Pb 2+ exposure support the idea that lead exposure can alter the lens biochemical homeostasis which may ultimately lead to loss of lens clarity with time. α-Crystallin, a major lens structural protein and molecular chaperone, undergoes various post-translational modifications upon aging which may contribute to decreased chaperone function and contribute to loss of lens clarity. This study evaluated the impact of 5 weeks of oral Pb 2+ exposure (peripheral Pb 2+ level ∼30 μg/dL) on the αA-crystallin protein profile of the lens from Fisher 344 rats. Decreases in relative protein spot intensity of more acidic forms of αA- and βA 4-crystallin and of truncated forms of αA-crystallin were noted. This data indicates that changes in post-translational processing of crystallins do occur in vivo following short courses of clinically relevant Pb 2+-exposure. In addition, organ culture of lenses from 4.5-month-old rats in 5 μM Pb 2+ resulted in opacities, demonstrating that lead is toxic to the lens and can induce a loss of lens clarity.

K-Ras-Independent Effects of the Farnesyl Transferase Inhibitor L-744,832 on Cyclin B1/Cdc2 Kinase Activity, G2/M Cell Cycle Progression and Apoptosis in Human Pancreatic Ductal Adenocarcinoma Cell

Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs). However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N) DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage -inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 µM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.

Processing of the gastrin precursor. Modulation of phosphorylated, sulfated, and amidated products.

Post-translational processing of the precursor for rat gastrin yields products that include peptides phosphorylated at Ser96, amidated at Phe92, and sulfated at Tyr87 or Tyr103. The phosphorylation site is immediately adjacent to the processing point that gives rise to the biologically active amidated gastrins. We have examined changes in post-translational processing which occur in gastrin cells from rats that are physiologically stimulated (by feeding) or unstimulated (by fasting). Peptides were identified using site-directed radioimmunoassays and chromatographic systems that resolve phosphorylated, amidated, and sulfated progastrin products, including intermediates generated prior to amidation (i.e. C-terminal glycine-extended variants). Assays for Phe92-amidated peptides and for the C-terminal tryptic fragment of progastrin indicated decreases in the total tissue concentrations of immunoreactive peptide with fasting; in contrast, the tissue concentrations of glycine-extended biosynthetic intermediates were similar in fasted and fed rats. Taken together the data suggest a relative failure in amidation mechanisms in unstimulated cells. The endopeptidase cleavage of progastrin was not influenced significantly by fasting. However, the phosphorylation of peptide products containing Ser96 was depressed significantly in fasted rats. The proportions of amidated peptides sulfated at Tyr87 were generally lower than their corresponding glycine-extended biosynthetic precursors, but in both cases the proportion of peptide in the sulfated form was lower than for peptides sulfated at Tyr103. Feeding did not change the sulfation of amidated heptadecapeptide gastrin or its glycine-extended variant. The results suggest that the mechanisms determining phosphorylation and amidation of progastrin-related peptides depend on the patterns of stimulation of gastrin cells. The observation that decreased phosphorylation is associated with a failure to produce active amidated products is consistent with a regulatory function for phosphorylation in gastrin production.

Modulation of posttranslational processing of gastrin precursor in dogs

The precursor of the acid-stimulating hormone gastrin is processed in pyloric antral gastrin cells by steps involving sulfation, phosphorylation, cleavage, and amidation. We describe here changes in posttranslational processing in dogs with a surgically excluded antrum; in the preparation we used there was an intact pylorus but antral mucosa was excluded from the normal influence of the luminal contents. Three to five months after the operation, basal plasma gastrin increased from 30.1 +/- 4.0 to 66.1 +/- 16.1 pmol/l, and concentrations of gastrin in the excluded mucosa were 9.23 +/- 1.75 compared with 3.2 +/- 0.56 nmol/g in control antral mucosa. Calculations based on the metabolic clearance rate and plasma and tissue gastrin concentrations suggest two-fold lower fractional release rates from the excluded G-cells compared with normal G-cells. Radioimmunoassay of tissue extracts using antisera specific for the extreme COOH-terminus of progastrin, for glycine-extended G-17, and for the COOH-terminus of G-17, combined with gel filtration and ion exchange chromatography, indicated normal endopeptidase cleavage of progastrin. However there was significantly reduced phosphorylation of the COOH-terminal tryptic fragment of progastrin, and there was also decreased conversion of Gly-extended intermediates to the biologically active COOH-terminally amidated forms of gastrin. Thus, in spite of hypergastrinaemia, the excluded antral mucosa showed evidence of decreased secretory rates associated with decreased progastrin phosphorylation and amidating enzyme activity. The results suggest that contact of antral mucosa with the luminal contents is able to modulate the posttranslational processing of progastrin and so determine the production of biologically active hormone.

Glucagon and Related Peptides in Fetal Rat Hypothalamusin Vivoandin Vitro*

Proglucagon-derived peptides are localized in pancreas, intestine, and the nervous system. We have examined the ontogeny of glucagon and related peptides in developing rat hypothalamus and have developed a fetal rat hypothalamic cell culture model to study the synthesis and secretion of these peptides in cells of neural origin. Fetal rat hypothalamus (19-21 day gestation) was found to contain glucagon-like immunoreactive (GLI) peptides including glucagon. The relative amounts of two of the GLI peptides (glicentin and oxyntomodulin) increased with development such that adult hypothalamus contained a predominance of these peptides over glucagon. The ratio of GLI peptides to glucagon increased from 2.6 +/- 0.5 in fetus to 46 +/- 11 in adult (P less than 0.001). When fetal rat hypothalamic cells (FRHC) were placed into primary culture for 7 days, the presence of neurons, glial cells, and glucagon-containing cells was detected by immunohistochemical staining. Analysis of proglucagon gene expression in FRHC cultures by Northern blotting demonstrated the presence of a single proglucagon messenger RNA (mRNA) transcript identical in size and sequence to that detected in fetal pancreas and intestine. RNase protection analysis of RNA from FRHC cultures, brainstem, and intestine confirmed that the proglucagon mRNA transcripts present in these three tissues were identical. Analysis of FRHC content of GLI peptides and immunoreactive glucagon demonstrated that peptide levels were not significantly different from those of whole fetal rat hypothalamus, and did not vary significantly throughout 2 weeks in culture. FRHC cultures were found to contain substantial amounts of glucagon after 1 week of culture. Release of the GLI peptides on day 7 of culture was increased 3-fold (P less than 0.001) by treatment of FRHC for 1 h with 5 mM (Bu)2cAMP. Rat hypothalamus therefore appears to undergo unique changes in posttranslational processing of proglucagon during development. Primary cultures of FRHC thus provide a promising in vitro model to study the molecular control of proglucagon biosynthesis and GLI peptide secretion in the brain.

Gonadal steroid and chronic morphine treatment do not change the posttranslational processing of β-endorphin in the rat brain

The present study examines whether two treatments known to induce refractoriness to exogenous morphine produce this desensitization through a change in the posttranslational processing of brain β-endorphin (β-End). The first experiment examined whether an ovarian steroid regimen which produces a transient desensitization of brain opiate receptor mechanisms alters β-End processing in the preoptic area (POA), medial basal hypothalamus (MBH), and brainstem (BS). The second experiment monitored the effects of morphine pellet treatment, known to produce morphine dependency, on immunoreactive β-End forms in the hypothalamus and periaquaductal gray area of the midbrain (PAG). The individual molecular forms of β-End were separated using ion exchange chromatography and collection fractions were quantitated for β-End immunoreactivity by RIA. The results show that regional differences occur in the posttranslational processing of β-End. In the hypothalamus, MBH and POA, β-End-(1–31) and its non-acetylated C- terminal cleavage products, β-End-(1–27) and β-End-(1–16) were the predominant forms of β-End. The PAG pools produced a β-End peptide elution profile similar to the hypothalamus, although small amounts of N-acetyl-β-End-(1–31) were also identified. The BS exhibited the least posttranslational processing of β-End; β-End-(1–31) was the primary product with smaller amounts of β-End-(1–27) and β-End-(1–26) observed. However, neither ovarian steroid treatment nor chronic morphine produced any changes in posttranslational processing of β-End or in total β-End concentration in any of the brain regions examined in these experiments. These data indicate that the refractoriness or tolerance to exogenous morphine associated with steroid or chronic morphine treatment cannot be explained by alterations in the biological activity of β-End resulting from the differential regulation of its posttranslational processing products.

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development: I. Adrenocorticotropin (ACTH) and α-melanotropins (α-MSHs)

We have studied the post-translational processing of POMC-derived peptides during fetal monkey development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Pituitary tissues obtained from fetal monkeys ranging from Gestational Day 50 to 155 were fractionated and analyzed for ACTH- and α-MSH-related peptides and compared to adult forms. Extracts of whole pituitary from Fetal Days 50 and 55 contained ACTH(1–39) and very small amounts of CLIP (corticotropin-like intermediate-lobe peptide; ACTH(18–39))-like immunoactivity. Acetylated α-MSHs were not detectable at Day 50. α-MSHs were barely detectable at Day 55. By Day 65, when pituitary lobes were separable, small amounts of des-, mono-, and diacetyl α-MSH were detectable in NIL extracts, but not in anterior lobe extracts. ACTH(1–39) levels were negligible when compared to increasing α-MSHs through Fetal Day 80 to 155 in the intermediate lobe. The CLIP immunoactivity was negligible in Day 80 and adult anterior lobe extracts. Thus, lobe-specific proteolytic processing of ACTH-related peptides was well established by midterm gestation. Marked increases of α- N- and α- N,O-acetylated forms of α-MSHs were detected during middle and late stage fetal development. Diacetyl α-MSH was the predominant form of α-MSH in adult NIL extracts. No acetylated α-MSHs were found in anterior lobe tissues, thus adult anterior lobe extracts contained almost exclusively ACTH(1–39). However adult NIL extracts contained two distinct forms of CLIP-related immunoactivity. Therefore changes in post-translational processing patterns of ACTH-related and α-MSH-related peptides continued to some extent, postnatally. These data indicate that marked changes in post-translational processing of POMC-derived ACTH-related products occur during the first half of monkey gestation.