Genetic events in cancer activate signalling pathways that alter cell metabolism. Clinical evidence has linked cell metabolism with cancer outcomes. Together, these observations have raised interest in targeting metabolic enzymes for cancer therapy, but they have also raised concerns that these therapies would have unacceptable effects on normal cells. However, some of the first cancer therapies that were developed target the specific metabolic needs of cancer cells and remain effective agents in the clinic today. Research into how changes in cell metabolism promote tumour growth has accelerated in recent years. This has refocused efforts to target metabolic dependencies of cancer cells as a selective anticancer strategy.

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Targeting cancer metabolism: a therapeutic window opens

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.

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Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

The fungal vacuole: composition, function, and biogenesis.

Cancer cells must rewire cellular metabolism to satisfy the demands of growth and proliferation. Although many of the metabolic alterations are largely similar to those in normal proliferating cells, they are aberrantly driven in cancer by a combination of genetic lesions and nongenetic factors such as the tumor microenvironment. However, a single model of altered tumor metabolism does not describe the sum of metabolic changes that can support cell growth. Instead, the diversity of such changes within the metabolic program of a cancer cell can dictate by what means proliferative rewiring is driven, and can also impart heterogeneity in the metabolic dependencies of the cell. A better understanding of this heterogeneity may enable the development and optimization of therapeutic strategies that target tumor metabolism.

Significance: Altered tumor metabolism is now a generally regarded hallmark of cancer. Nevertheless, the recognition of metabolic heterogeneity in cancer is becoming clearer as a result of advancements in several tools used to interrogate metabolic rewiring and dependencies. Deciphering this context-dependent heterogeneity will supplement our current understanding of tumor metabolism and may yield promising therapeutic and diagnostic utilities. Cancer Discov; 2(10); 881–98. ©2012 AACR .

https://cancerdiscovery.aacrjournals.org/content/candisc/2/10/881.full.pdf

Cancer Cell Metabolism: One Hallmark, Many Faces

Chemistry of polyethylene glycol conjugates with biologically active molecules

Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90° light scattering data in circular dichroism (CD) spectroscopic experiments. Since the measurements of turbidity and 90° light scattering can be done simultaneously with far- or near-UV CD data collection, they require no additional time or sample and can be directly correlated with the protein conformational changes monitored by CD. The results can provide useful insights into the origins of irreversible conformational changes and test the linkage between protein unfolding or misfolding and aggregation in various macromolecular systems, including globular proteins and protein–lipid complexes described in this study, as well as a wide range of amyloid-forming proteins and peptides.

/pdf/monitoring-protein-aggregation-during-thermal-unfolding-in-49u4vrb4rl.pdf

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments.

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248.

The amino acid sequence and tertiary structure of Wolinella succinogenesl-asparaginase were determined, and were compared with the structures of other type-II bacterial l-asparaginases. Each chain of this homotetrameric enzyme consists of 330 residues. The amino acid sequence is 40–50% identical to the sequences of related proteins from other bacterial sources, and all residues previously shown to be crucial for the catalytic action of these enzymes are identical. Differences between the amino acid sequence of W succinogenesl-asparaginase and that of related enzymes are discussed in terms of the possible influence on the substrate specificity. The overall fold of the protein subunit is almost identical to that observed for other l-asparaginases. Two fragments in each subunit, a very highly flexible loop (?20 amino acids) that forms part of the active site, and the N-terminus (two amino acids), are not defined in the structure. The orientation of Thr14, a residue probably involved in the catalytic activity, indicates the absence of ligand in the active-site pocket. The rigid part of the active site, which includes the asparaginase triad Thr93-Lys166-Asp94, is structurally very highly conserved with equivalent regions found in other type-II bacterial l-asparaginases.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1996.0201t.x

Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase.

Direct curve-fitting is uncontested as the method of choice for evaluating kinetic experiments. If carried out by computer, the numerical solution of such problems is superior to customary graphical procedures in any respect. However, most programs available to date, cannot be executed by small systems. We now describe a BASIC program for desk-top microcomputers that performs linear and non-linear regression analysis of kinetic data. It may be directly applied to a number of problems common in enzyme kinetics, and is easily modified to handle further ones. The performance of the program is illustrated by some typical applications; in addition, fundamental statistical methods are discussed that allow a critical examination of the results obtained.

Microcomputers in enzymology. A versatile BASIC computer program for analyzing kinetic data

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2896%2900660-6

Klaus-Heinrich Röhm

Papers

Monitoring protein aggregation during thermal unfolding in circular dichroism experiments.

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248.

Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase.

Microcomputers in enzymology. A versatile BASIC computer program for analyzing kinetic data

A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant.