Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2010; 107(29):12872-6. DOI: 10.1073/pnas.1008451107
Source: PubMed


Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.

Download full-text


Available from: John W Tomsho
  • Source
    • "It seems that the ratio of the accumulating S-Ado and SAICAr in body fluids is not predictive of phenotype severity; rather, it may be secondary to the degree of the patient's development (i.e., to the age of the patient at the time of a sample collection) (Zikanova et al 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adenylosuccinate lyase ADSL) deficiency is a defect of purine metabolism affecting purinosome assembly and reducing metabolite fluxes through purine de novo synthesis and purine nucleotide recycling pathways. Biochemically this defect manifests by the presence in the biologic fluids of two dephosphorylated substrates of ADSL enzyme: succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado). More than 80 individuals with ADSL deficiency have been identified, but incidence of the disease remains unknown. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The fatal neonatal form has onset from birth and presents with fatal neonatal encephalopathy with a lack of spontaneous movement, respiratory failure, and intractable seizures resulting in early death within the first weeks of life. Patients with type I (severe form) present with a purely neurologic clinical picture characterized by severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features. A more slowly progressing form has also been described (type II, moderate or mild form), as having later onset, usually within the first years of life, slight to moderate psychomotor retardation and transient contact disturbances. Diagnosis is facilitated by demonstration of SAICAr and S-Ado in extracellular fluids such as plasma, cerebrospinal fluid and/or followed by genomic and/or cDNA sequencing and characterization of mutant proteins. Over 50 ADSL mutations have been identified and their effects on protein biogenesis, structural stability and activity as well as on purinosome assembly were characterized. To date there is no specific and effective therapy for ADSL deficiency.
    Full-text · Article · Aug 2014 · Journal of Inherited Metabolic Disease
  • Source
    • "Strikingly, recent visual screens of the yeast green fluorescent protein (GFP) strain collection reveal that multiple metabolic enzymes self-assemble into filaments, arguing that this mode of regulation could play a role in the control of many biosynthetic pathways (Narayanaswamy et al., 2009; Noree et al., 2010). However, although the pace at which novel cytoplasmic structures are being identified continues to accelerate, little is known about how specific enzyme regulatory mechanisms impact the large cytoplasmic structures they form. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ability of enzymes to assemble into visible supramolecular complexes is now appreciated to be a widespread phenomenon. Such complexes have been hypothesized to play a number of roles, however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure-function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments are comprised of inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity.
    Full-text · Article · Jun 2014 · Molecular Biology of the Cell
  • Source
    • "Hallmark proteins such as alpha-synuclein=TPPP=p25 and beta-amyloid=hyperphosphorylated tau are enriched in the pathological inclusions in the case of well-established neurological disorders, PD and AD, respectively [Kovacs et al., 2004; Irvine et al., 2008; Orosz et al., 2009]. The etiology of these diseases is a complex process; different mechanisms acting under pathological conditions [Beal, 2000], some of them related to the dysfunction of cytoskeleton dynamics, contribute to the development of neurodegenerative diseases [Parisiadou and Cai, 2010; Esteves et al., 2011]. One of the characteristics of AD is the formation of neurofibrillary tangles consisting of hyperphosphorylated tau protein [Marx, 2007]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The sensing, integrating and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting Tubulin Polymerization Promoting Protein, TPPP/p25. TPPP/p25 is a Microtubule Associated Protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The non-physiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Nov 2013 · Cytoskeleton
Show more