David A. Maltby

Global landscape of HIV-human protein complexes

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host’s cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV–human protein–protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

O-Linked N-Acetylglucosamine Proteomics of Postsynaptic Density Preparations Using Lectin Weak Affinity Chromatography and Mass Spectrometry

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. β-Elimination/Michael addition, MS3 on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including “PVST” and a novel “TTA” motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.

Promiscuous Aggregate-Based Inhibitors Promote Enzyme Unfolding

One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen−deuterium exchange mass spectrometry. For AmpC β-lactamase, binding to aggregates of the small molecule rottlerin increased the deuterium exchange of all 10 reproducibly detectable peptides, which covered 41% of the sequence of β-lactamase. This suggested a global increase in proton accessibility upon aggregate binding, consistent with denaturation. We then investigated whether enzyme−aggregate complexes were more susceptible to proteolysis than uninhibited enzyme. For five aggregators, trypsin degradation of β-lactamase increased substantially when β-lactamase was inhibited by aggregates, whereas uninhibited enzyme was generally stable to digestion. Combined, these results suggest that the mechanism of action of aggregate-based inhibitors proceeds via partial protein unfolding when bound to an aggregate particle.

Recognition of medium-chain acyl-CoA dehydrogenase deficiency in asymptomatic siblings of children dying of sudden infant death or Reye-like syndromes*

The medium-chain acyl-CoA dehydrogenase (MCAD) deficiency of mitochondrial beta oxidation has been identified in two asymptomatic siblings in a family in which two previous deaths had been recorded, one attributed to sudden infant death syndrome and the other to Reye syndrome. Recognition of this disorder in one of the deceased and in the surviving siblings was accomplished by detection of a diagnostic metabolite, octanoylcarnitine, using a new mass spectrometric technique. This resulted in early treatment with L-carnitine supplement in the survivors, which should prevent metabolic deterioration. Further studies suggest that breast-feeding may be protective for infants with MCAD deficiency. Families with children who have had Reye syndrome or in which sudden infant death has occurred are at risk for MCAD deficiency. We suggest that survivors and asymptomatic siblings should be tested for this treatable disorder.

An antibiotic factory caught in action.

The synthesis of aromatic polyketides, such as actinorhodin, tetracycline and doxorubicin, begins with the formation of a polyketide chain. In type II polyketide synthases (PKSs), chains are polymerized by the heterodimeric ketosynthase–chain length factor (KS-CLF). Here we present the 2.0-Å structure of the actinorhodin KS-CLF, which shows polyketides being elongated inside an amphipathic tunnel ∼17 Å in length at the heterodimer interface. The structure resolves many of the questions about the roles of KS and CLF. Although CLF regulates chain length, it does not have an active site; KS must catalyze both chain initiation and elongation. We provide evidence that the first cyclization of the polyketide occurs within the KS-CLF tunnel. The mechanistic details of this central PKS polymerase could guide biosynthetic chemists in designing new pharmaceuticals and polymers.

Identification and molecular cloning of a neuropeptide Y homolog that produces prolonged inhibition in aplysia neurons

The neuroendocrine bag cell neurons of the marine mollusk Aplysia produce prolonged inhibition that lasts for more than 2 hr. We purified a peptide from the abdominal ganglion that mimics this inhibition. Mass spectrometry and microsequence analysis indicate that the peptide is 40 aa long and is amidated at its carboxyl terminus. It is highly homologous to vertebrate neuropeptide Y (NPY) and other members of the pancreatic polypeptide family. As determined from cloned cDNA, the gene coding for the precursor protein shares a common structural organization with genes encoding precursors of the vertebrate family. The peptides may therefore have arisen from a common ancestral gene. Bag cell neurons are immunoreactive for Aplysia NPY, and Northern blot analysis indicates that as with its vertebrate counterparts, the peptide is abundantly expressed in the CNS. This suggests that peptides related to NPY may have important functions in the nervous system of Aplysia as well as in other invertebrates.

L-carnitine enhances excretion of propionyl coenzyme A as propionylcarnitine in propionic acidemia.

Treatment with L-carnitine greatly enhanced the formation and excretion of short-chain acylcarnitines in three patients with propionic acidemia and in three normal controls. The use of fast atom bombardment mass spectrometry and linked scanning at constant magnetic (B) to electric (E) field ratio identified the acylcarnitine as propionylcarnitine in patients with propionic acidemia. The normal children excreted mostly acetylcarnitine. Propionic acidemia and other organic acidurias are characterized by the intramitochondrial accumulation of short-chain acyl-Coenzyme A (CoA) compounds. The substrate specificity of the carnitine acetyltransferase enzyme and its steady state nature appears to facilitate elimination of propionyl groups while restoring the acyl-CoA:free CoA ratio in the mitochondrion. We suggest that L-carnitine may be a useful therapeutic approach for elimination of toxic acyl CoA compounds in several of these disorders.

Diagnostic and Therapeutic Implications of Medium-Chain Acylcarnitines in the Medium-Chain Acyl-CoA Dehydrogenase Deficiency

ABSTRACT: The medium-chain acyl-coA dehydrogenase deficiency is one of several metabolic disorders presenting clinically as Reye syndrome. Evidence is presented for a characteristic organic aciduria that distinguishes this disorder from Reye syndrome and other masqueraders characterized by dicarboxylic aciduria. The key metabolites, suberylglycine and hexanoylglycine, are excreted in high concentration only when the patients are acutely ill. More significantly, using novel techniques in mass spectrometry, the medium-chain defect is shown to be characterized by excretion of specific medium-chain acylcarnitines, mostly octanoylcarnitine, without significant excretion of a normal metabolite, acetylcarnitine, in four patients with documented enzyme deficiency. Similar studies on the urine of two patients reported with Reye-like syndromes of unidentified etiology have suggested the retrospective diagnosis of medium-chain acyl-coA dehydrogenase deficiency. Administration of L-carnitine to medium-chain acyl-coA dehydrogenase deficiency patients resulted in the enhanced excretion of medium-chain acylcarnitines. Octanoylcarnitine is prominent in the urine both prior to and following L-carnitine supplementation. The detection of this metabolite as liberated octanoic acid, following ion-exchange chromatographic purification and mild alkaline hydrolysis, provides a straightforward diagnostic procedure for recognition of this disorder without subjecting patients to the significant risk of fasting. In view of the carnitine deficiency and the demonstrated ability to excrete the toxic mediumchain acyl-coA compounds as acylcarnitines, a combined therapy of reduced dietary fat and L-carnitine supplementation (25 mg/kg/6 h) has been devised and applied with positive outcome in two new cases.

Inflammatory Stimuli Regulate Caspase Substrate Profiles

The inflammatory caspases, human caspases-1, -4, and -5, proteolytically modulate diverse physiological outcomes in response to proinflammatory signals. Surprisingly, only a few substrates are known for these enzymes, including other caspases and the interleukin-1 family of cytokines. To more comprehensively characterize inflammatory caspase substrates, we combined an enzymatic N-terminal enrichment method with mass spectrometry-based proteomics to identify newly cleaved proteins. Analysis of THP-1 monocytic cell lysates treated with recombinant purified caspases identified 82 putative caspase-1 substrates, three putative caspase-4 substrates, and no substrates for caspase-5. By contrast, inflammatory caspases activated in THP-1 cells by mimics of gout (monosodium urate), bacterial infection (lipopolysaccharide and ATP), or viral infection (poly(dA·dT)) were found to cleave only 27, 16, and 22 substrates, respectively. Quantitative stable isotope labeling with amino acids in cell culture (SILAC) comparison of these three inflammatory stimuli showed that they induced largely overlapping substrate profiles but different extents of proteolysis. Interestingly, only half of the cleavages found in response to proinflammatory stimuli were contained within our set of 82 in vitro cleavage sites. These data provide the most comprehensive set of caspase-1-cleaved products reported to date and indicate that caspases-4 and -5 have far fewer substrates. Comparisons between the in vitro and in vivo data highlight the importance of localization in regulating inflammatory caspase activity. Finally, our data suggest that inducers of inflammation may subtly alter caspase-1 substrate profiles.

L-carnitine therapy in isovaleric acidemia.

Isovaleric acidemia, resulting from isovaleryl-coenzyme A dehydrogenase deficiency, is associated with marked reduction of free carnitine in both plasma and urine. Fast atom bombardment-mass spectrometry, hydrolysis, and gas chromatography/mass spectrometry have unequivocally identified the existence of isovalerylcarnitine, a new metabolite specific for this disorder. Administration of equimolar amounts of glycine or L-carnitine separately with leucine demonstrated that isovaleryl-coenzyme A is removed by supplemental L-carnitine in the form of isovalerylcarnitine as effectively as it is by glycine, in the form of isovalerylglycine. When L-carnitine is given alone, excretion of isovalerylglycine decreases in preference to enhanced excretion of isovalerylcarnitine and hippurate. Treatment with L-carnitine alone has proven effective in preventing further hospitalizations in a patient with this genetic disorder.