Judit Ovádi

Macromolecular compartmentation and channeling.

One of the accepted characterizations of the living state is that it is complex to an extraordinary degree. Since our current understanding of the living condition is minimal and fragmentary, it is not surprising that our first descriptions are simplistic. However, in certain areas of metabolism, especially those that have been amenable to experimentation for the longest period of time, the simplistic explanations have been the most difficult to revise. For example, current texts of general biochemistry still view metabolism as occurring by a series of independent enzymes dispersed in a uniform aqueous environment. This notion has been shown to be deeply flawed by both experimental and theoretical considerations. Thus, there is ample evidence that, in many metabolic pathways, specific interactions between sequential enzymes occur as static and/or dynamic complexes. In addition, reversible interactions of enzymes with structural proteins and membranes is a common occurrence. The interactions of enzymes give rise to a higher level of complexity that must be accounted for when one wishes to understand the regulation of metabolism. One of the phenomena that occurs because of sequential enzyme interactions is the process of channeling. This article discusses enzyme interactions and channeling and summarizes experimental and theoretical results from a few well-studied examples.

On the origin of intracellular compartmentation and organized metabolic systems.

The history of the development of the ideas and research of organized metabolic systems during last three decades is shortly reviewed. The cell cytoplasm is crowded with solutes, soluble macromolecules such as enzymes, nucleic acids, structural proteins and membranes. The high protein density within the large compartments of the cells predominantly determines the major characteristics of cellular environment such as viscosity, diffusion and inhomogeneity. The fact that the solvent viscosity of cytoplasm is not substantially different from the water is explained by intracellular structural heterogeneity: the intrinsic macromolecular density is relatively low within the interstitial voids in the cell because many soluble enzymes are apparently integral parts of the insoluble cytomatrix and are not distributed homogeneously. The molecular crowding and sieving restrict the mobility of very large solutes, binding severely restrict the mobility of smaller solutes. One of consequence of molecular crowding and hindered diffusion is the need to compartmentalize metabolic pathway to overcome diffusive barriers. Although the movement of small molecules is slowed down in the cytoplasm, the metabolism can successfully proceed and even be facilitated by metabolite channeling which directly transfers the intermediate from one enzyme to an adjacent enzyme without the need of free aqueous-phase diffusion. The enhanced probability for intermediates to be transfered from one active site to the other by sequential enzymes requires stable or transient interactions of the relevant enzymes, which associate physically in non-dissociable, static multienzyme complexes – metabolones, particles containing enzymes of a part or whole metabolic systems. Therefore, within the living cell the metabolism depends on the structural organization of enzymes forming microcompartments. Since cells contain many compartments and microenvironments, the measurement of the concentration of metabolites in whole cells or tissues gives an average cellular concentration and not that which is actually sensed by the active site of a specific enzyme. Thus, the microcompartmentation could provide a mechanism which can control metabolic pathways. Independently and in parallel to the developments described above, the ideas of compartmentation came into existence from the necessity to explain important physiological phenomena, in particular in heart research and in cardiac electrophysiology. These phenomena demonstrated the physiological importance of the biophysical and biochemical mechanisms described in this review.

Nuclear Translocation of the Tumor Marker Pyruvate Kinase M2 Induces Programmed Cell Death

Cancer cells often fail to respond to stimuli that normally activate their intrinsic apoptotic machinery. Moreover, they are able to adapt to hypoxia by changing their glycolytic rate. Pyruvate kinase (PK) is a rate-limiting enzyme in glycolysis that is converted to a less active dimer form of PKM2 isoenzyme during oncogenesis. Here, we show that both somatostatin and the structural analogue TT-232 interact with the PKM subtype. We further show that the PKM2 is translocated to the nucleus in response to TT-232 and different apoptotic agents. Nuclear translocation of PKM2 is sufficient to induce cell death that is caspase independent, isoform specific, and independent of its enzymatic activity. These results show that the tumor marker PKM2 plays a general role in caspase-independent cell death of tumor cells and thereby defines this glycolytic enzyme as a novel target for cancer therapy development.

Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies

The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinsons (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Picks disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimers disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

Enzyme-enzyme interactions and their metabolic role

There are continuing reports on the existence of complexes of sequential metabolic enzymes. New techniques for their detection have been described and include affinity electrophoresis and the use of anti‐idiotypic antibodies. Channeling of substrates has been reported for several systems as well as direct substrate transfer through dynamic enzyme associations. Kinetic parameters of metabolic control of organized systems have been formulated and tested in several systems. These recent results are expanding our understanding of metabolic processes and their control.

TPPP/p25 promotes tubulin assemblies and blocks mitotic spindle formation.

Recently, we isolated from bovine brain a protein, TPPP/p25 and identified as p25, a brain-specific protein that induced aberrant tubulin assemblies. The primary sequence of this protein differs from that of other proteins identified so far; however, it shows high homology with p25-like hypothetical proteins sought via blast. Here, we characterized the binding of TPPP/p25 to tubulin by means of surface plasmon resonance; the kinetic parameters are as follows: kon, 2.4 × 104 M–1·s–1; koff, 5.4 × 10–3 s–1; and Kd, 2.3 × 10–7 M. This protein at substoichometric concentration promotes the polymerization of tubulin into double-walled tubules and polymorphic aggregates or bundles paclitaxel-stabilized microtubules as judged by quantitative data of electron and atomic force microscopies. Injection of bovine TPPP/p25 into cleavage Drosophila embryos expressing tubulin–GFP fusion protein reveals that TPPP/p25 inhibits mitotic spindle assembly and nuclear envelope breakdown without affecting other cellular events like centrosome replication and separation, microtubule nucleation by the centrosomes, and nuclear growth. GTP counteracts TPPP/p25 both in vitro and in vivo.

Triosephosphate isomerase deficiency: new insights into an enigmatic disease

The triosephosphate isomerase (TPI) functions at a metabolic cross-road ensuring the rapid equilibration of the triosephosphates produced by aldolase in glycolysis, which is interconnected to lipid metabolism, to glycerol-3-phosphate shuttle and to the pentose phosphate pathway. The enzyme is a stable homodimer, which is catalytically active only in its dimeric form. TPI deficiency is an autosomal recessive multisystem genetic disease coupled with hemolytic anemia and neurological disorder frequently leading to death in early childhood. Various genetic mutations of this enzyme have been identified; the mutations result in decrease in the catalytic activity and/or the dissociation of the dimers into inactive monomers. The impairment of TPI activity apparently does not affect the energy metabolism at system level; however, it results in accumulation of dihydroxyacetone phosphate followed by its chemical conversion into the toxic methylglyoxal, leading to the formation of advanced glycation end products. By now, the research on this disease seems to enter a progressive stage by adapting new model systems such as Drosophila, yeast strains and TPI-deficient mouse, which have complemented the results obtained by prediction and experiments with recombinant proteins or erythrocytes, and added novel data concerning the complexity of the intracellular behavior of mutant TPIs. This paper reviews the recent studies on the structural and catalytic changes caused by mutation and/or nitrotyrosination of the isomerase leading to the formation of an aggregation-prone protein, a characteristic of conformational disorders.

Tubulin polymerization-promoting protein (TPPP/p25) is critical for oligodendrocyte differentiation.

TPPP/p25, a recently identified tubulin polymerization‐promoting protein (TPPP), is expressed mainly in myelinating oligodendrocytes of the CNS. Here, we show that TPPP/p25 is strongly upregulated during the differentiation of primary oligodendrocyte cells as well as the CG‐4 cell line. The microRNA expression profile of CG‐4 cells before and after induction of differentiation was established and revealed differential regulation of a limited subset of microRNAs. miR‐206, a microRNA predicted to target TPPP/p25, was not detected in oligodendrocytes. Overexpression of miR‐206 led to downregulation of TPPP/p25 resulting in inhibition of differentiation. Transfection of siRNAs against TPPP/p25 also inhibited cell differentiation and promoted cell proliferation, providing evidence for an important role of TPPP/p25 during oligodendrogenesis. These results support an essential role for TPPP/p25 in oligodendrocyte differentiation likely via rearrangement of the microtubule system during the process elongation prior to the onset of myelination.

Triosephosphate isomerase deficiency: Facts and doubts

Many glycolytic enzymopathies have been described that manifest clinically as chronic hemolytic anemia. One of these, triosephosphate isomerase (TPI) deficiency, is unique among the glycolytic enzyme defects since it is associated with progressive neurological dysfunction and frequently with childhood death. The physiological function of TPI is to adjust the rapid equilibrium between dihydroxyacetone phosphate and glyceraldehyde‐3‐phosphate produced by aldolase in glycolysis, which is interconnected to the pentose phosphate pathway and to lipid metabolism via triosephosphates. The TPI gene is well characterized; structure and function studies suggest that instability of the isomerase due to different mutations of the enzyme may underlie the observed reduced catalytic activity. Patients with various inherited mutations have been identified. The most abundant mutation is a Glu104Asp missense mutation that is found in homozygotes and compound heterozygotes. Two germ‐line identical Hungarian compound heterozygote brothers with distinct phenotypes question the exclusive role of the inherited mutations in the etiology of neurodegeneration. This paper: (i) reviews our present understanding of TPI mutation‐induced structural alterations and their pathological consequences, (ii) summarizes the consequences of TPI impairment in the Hungarian case at local and system levels, and (iii) raises critical questions regarding the exclusive role of TPI mutations in the development of this human disease. iubmb Life, 58: 703‐715, 2006

Selective Sirt2 inhibition by ligand-induced rearrangement of the active site

Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.