Polyubiquitin chains: functions, structures, and mechanisms

Ubiquitin is a highly conserved 76-amino acid polypeptide that is found throughout the eukaryotic kingdom. The covalent conjugation of ubiquitin (often in the form of a polymer) to substrates governs a variety of biological processes ranging from proteolysis to DNA damage tolerance. The functional flexibility of this post-translational modification has its roots in the existence of a large number of ubiquitinating enzymes that catalyze the formation of distinct ubiquitin polymers, which in turn encode different signals. This review summarizes recent advances in the field with an emphasis on the non-canonical functions of polyubiquitination. We also discuss the potential mechanism of chain linkage specification as well as how structural disparity in ubiquitin polymers may be distinguished by ubiquitin receptors to translate the versatile ubiquitin signals into various cellular functions.     

Eukaryotic DNA damage checkpoint activation in response to double-strand breaks

Double-strand breaks (DSBs) are the most detrimental form of DNA damage. Failure to repair these cytotoxic lesions can result in genome rearrangements conducive to the development of many diseases, including cancer. The DNA damage response (DDR) ensures the rapid detection and repair of DSBs in order to maintain genome integrity. Central to the DDR are the DNA damage checkpoints. When activated by DNA damage, these sophisticated surveillance mechanisms induce transient cell cycle arrests, allowing sufficient time for DNA repair. Since the term “checkpoint” was coined over 20 years ago, our understanding of the molecular mechanisms governing the DNA damage checkpoint has advanced significantly. These pathways are highly conserved from yeast to humans. Thus, significant findings in yeast may be extrapolated to vertebrates, greatly facilitating the molecular dissection of these complex regulatory networks. This review focuses on the cellular response to DSBs in Saccharomyces cerevisiae, providing a comprehensive overview of how these signalling pathways function to orchestrate the cellular response to DNA damage and preserve genome stability in eukaryotic cells.     

Synonymous codons,ribosome speed,and eukaryotic gene expression regulation

Quantitative control of gene expression occurs at multiple levels, including the level of translation. Within the overall process of translation, most identified regulatory processes impinge on the initiation phase. However, recent studies have revealed that the elongation phase can also regulate translation if elongation and initiation occur with specific, not mutually compatible rate parameters. Translation elongation then limits the overall amount of protein that can be made from an mRNA. Several recently discovered control mechanisms of biological pathways are based on such elongation control. Here, we review the molecular mechanisms that determine ribosome speed in eukaryotic organisms, and discuss under which conditions ribosome speed can become the controlling parameter of gene expression levels.     

Endonuclease V: an unusual enzyme for repair of DNA deamination

Endonuclease V (endo V) was first discovered as the fifth endonuclease in Escherichia coli in 1977 and later rediscovered as a deoxyinosine 3′ endonuclease. Decades of biochemical and genetic investigations have accumulated rich information on its role as a DNA repair enzyme for the removal of deaminated bases. Structural and biochemical analyses have offered invaluable insights on its recognition capacity, catalytic mechanism, and multitude of enzymatic activities. The roles of endo V in genome maintenance have been validated in both prokaryotic and eukaryotic organisms. The ubiquitous nature of endo V in the three domains of life: Bacteria, Archaea, and Eukaryotes, indicates its existence in the early evolutionary stage of cellular life. The application of endo V in mutation detection and DNA manipulation underscores its value beyond cellular DNA repair. This review is intended to provide a comprehensive account of the historic aspects, biochemical, structural biological, genetic and biotechnological studies of this unusual DNA repair enzyme.     

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein

Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.     

Autophagy: molecular mechanisms,physiological functions and relevance in human pathology

Autophagy is a degradative mechanism mainly involved in the recycling and turnover of cytoplasmic constituents from eukaryotic cells. Over the last years, yeast genetic screens have considerably increased our knowledge about the molecular mechanisms of autophagy, and a number of genes involved in fundamental steps of the autophagic pathway have been identified. Most of these autophagy genes are present in higher eukaryotes indicating that this process has been evolutionarily conserved. In yeast, autophagy is mainly involved in adaptation to starvation, but in multicellular organisms this route has emerged as a multifunctional pathway involved in a variety of additional processes such as programmed cell death, removal of damaged organelles and development of different tissue-specific functions. Furthermore, autophagy is associated with a growing number of pathological conditions, including cancer, myopathies and neurodegenerative disorders. The physiological and pathological roles of autophagy, as well as the molecular mechanisms underlying this multifunctional pathway, are discussed in this review.Received 12 January 2004; received after revision 29 January 2004; accepted 4 February 2004     

Functions of actin in endocytosis

Endocytosis is a fundamental eukaryotic process required for remodelling plasma-membrane lipids and protein to ensure appropriate membrane composition. Increasing evidence from a number of cell types reveals that actin plays an active, and often essential, role at key endocytic stages. Much of our current mechanistic understanding of the endocytic process has come from studies in budding yeast and has been facilitated by yeast’s genetic amenability and by technological advances in live cell imaging. While endocytosis in metazoans is likely to be subject to a greater array of regulatory signals, recent reports indicate that spatiotemporal aspects of vesicle formation requiring actin are likely to be conserved across eukaryotic evolution. In this review we focus on the ‘modular’ model of endocytosis in yeast before highlighting comparisons with other cell types. Our discussion is limited to endocytosis involving clathrin as other types of endocytosis have not been demonstrated in yeast.     

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.     

The CLE family of plant polypeptide signaling molecules

Polypeptide ligands have long been recognized as primary signaling molecules in diverse physiological processes in animal systems. Recent studies in plants have provided major breakthroughs with the discovery that small polypeptides are also involved in many plant biological processes, indicating that the use of polypeptides as signaling molecules in cell-to-cell communication is evolutionarily conserved. The CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) proteins are currently the best understood family of small polypeptides in plants. The recent isolation of MCLV3 from Arabidopsis and TDIF from a Zinnia cell culture system indicates that biologically active CLE polypeptides are produced by post-translational proteolysis and modification, similar to peptide hormone production in animals and yeast. Here, we review exciting discoveries involving the identification of the CLE proteins and their functions in various aspects of plant development, including restriction of stem cell accumulation by CLV3 and inhibition of xylem differentiation by TDIF.     

La FAM fatale: USP9X in development and disease

Deubiquitylating enzymes (DUBs), act downstream of ubiquitylation. As such, these post-post-translational modifiers function as the final arbitrators of a protein substrate’s ubiquitylation status, thus regulating its fate. In most instances, DUBs moderate the absolute level of a substrate, its locality or activity, rather than being an “all-or-none” phenomenon. Yet, disruption of this quantitative regulation can produce dramatic qualitative differences. The ubiquitin-specific protease 9X (USP9X/FAM) is a substrate-specific DUB, which displays an extraordinarily high level of sequence conservation from Drosophila to mammals. It is primarily the recent revelations of USP9X’s pivotal role in human cancers, both as oncogene or tumour suppressor, in developmental disorders including intellectual disability, epilepsy, autism and developmental delay that has led to a subsequent re-examination of its molecular and cellular functions. Results from experimental animal models have implicated USP9X in neurodegeneration, including Parkinson’s and Alzheimer’s disease, as well as autoimmune diseases. In this review, we describe the current and accumulated knowledge on the molecular, cellular and developmental aspects of USP9X function within the context of the biological consequences during normal development and disease.     

Structure of the gap junction channel and its implications for its biological functions

Gap junctions consist of arrays of intercellular channels composed of integral membrane proteins called connexin in vertebrates. Gap junction channels regulate the passage of ions and biological molecules between adjacent cells and, therefore, are critically important in many biological activities, including development, differentiation, neural activity, and immune response. Mutations in connexin genes are associated with several human diseases, such as neurodegenerative disease, skin disease, deafness, and developmental abnormalities. The activity of gap junction channels is regulated by the membrane voltage, intracellular microenvironment, interaction with other proteins, and phosphorylation. Each connexin channel has its own property for conductance and molecular permeability. A number of studies have tried to reveal the molecular architecture of the channel pore that should confer the connexin-specific permeability/selectivity properties and molecular basis for the gating and regulation. In this review, we give an overview of structural studies and describe the structural and functional relationship of gap junction channels.     

Aging and TOR: interwoven in the fabric of life

Longstanding results with calorie and growth factor restriction plus recent results with the first interventional drug suggest that retarding the pace of aging to improve the quality of life of older people is at hand. The biological system targeted by these approaches is the target of rapamycin (TOR), which is central for cellular responses to a variety of stimuli including stressors, growth factors, and nutrients and energy states. That the life-extending response to reducing its activity is highly conserved from yeast to mammals is consistent with the evolution of aging as a strategy to preserve reproductive potential of young cells and animals.     

From immune response to cancer: a spot on the low molecular weight protein tyrosine phosphatase

Reversible tyrosine phosphorylation is a key posttranslational regulatory modification of proteins in all eukaryotic cells in normal and pathological processes. Recently a pivotal janus-faced biological role of the low molecular weight protein tyrosine phosphatase (LMWPTP) has become clear. On the one hand this enzyme is important in facilitating appropriate immune responses towards infectious agents, on the other hand it mediates exaggerated inflammatory responses toward innocuous stimuli. The evidence that LMWPTP plays a role in oncological processes has added a promising novel angle. In this review we shall focus on the regulation of LMWPTP enzymatic activity of signaling pathways of different immunological cells, the relation between genetic polymorphism of LMWPTP and predisposition to some type of inflammatory disorders and the contribution of this enzyme to cancer cell onset, growth and migration. Therefore, the LMWPTP is an interesting target for pharmacological intervention, thus modifying both inappropriate cellular immune responses and cancer cell aggressiveness. Received 15 August 2008; received after revision 06 October 2008; accepted 14 October 2008     

Vacuolar H+-ATPase: From mammals to yeast and back

Vacuolar H⁺-adenosine triphosphatase (V-ATPase) is composed of distinct catalytic (V₁) and membrane (V₀) sectors containing several subunits. The biochemistry of the enzyme was mainly studied in organelles from mammalian cells such as chromaffin granules and clathrin-coated vesicles. Subsequently, mammalian cDNAs and yeast genes encoding subunits of V-ATPase were cloned and sequenced. The sequence information revealed the relation between V- and F-ATPases that evolved from a common ancestor. The isolation of yeast genes encoding subunits of V-ATPase opened an avenue for molecular biology studies of the enzyme. Because V-ATPase is present in every known eukaryotic cell and provides energy for vital transport systems, it was anticipated that disruption of genes encoding V-ATPase subunits would be lethal. Fortunately, yeast cells can survive the absence of V-ATPase by drinking the acidic medium. So far only yeast cells have been shown to be viable without an active V-ATPase. In contrast to yeast, mammalian cells may have more than one gene encoding each of the subunits of the enzyme. Some of these genes encode tissue- and/or organelle-specific subunits. Expression of these specific cDNAs in yeast cells may reveal their unique functions in mammalian cells. Following the route from mammals to yeast and back may prove useful in the study of many other complicated processes.