Calcium-dependent proteases: an enzyme system active at cellular membranes?

Proteases having a neutral pH optimum and an absolute requirement for calcium ion are found in virtually all mammalian cells. Association of calcium-dependent proteases and a specific inhibitor protein with biological membranes seems to be an important regulatory feature of this proteolytic system, and it is likely that membranes are preferred sites for calcium-dependent protease action. Several recent hypotheses for the physiological function of calcium-dependent proteolysis are consistent with a membrane-associated protease action. Calcium-dependent proteases may participate in cell membrane fusion: the proteolysis of membrane proteins, which is required for the efficient fusion of erythrocytes, may be catalyzed by these enzymes. There is also evidence for the involvement of calcium-dependent proteolysis in postsynaptic membrane remodeling in the hippocampus after long-term potentiation. Although the relationship of the proteolysis to synaptic function is not known, it could have important physiological or pathophysiological consequences. Finally, it has recently been suggested that calcium-dependent proteolysis may be a physiologically significant mechanism for activating membrane-associated protein kinase C after exposure of some cell types to phorbol esters or other mitogens. Further pursuit of these hypotheses may reveal a novel role for intracellular calcium-regulated proteolysis in membrane-associated cell functions.     

Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes

Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few 'druggable' classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.     

Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!

Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.     

Untangling structure–function relationships in the rhomboid family of intramembrane proteases

Rhomboid proteases are a family of integral membrane proteins that have been implicated in critical regulatory roles in a wide array of cellular processes and signaling events. The determination of crystal structures of the prokaryotic rhomboid GlpG from Escherichia coli and Haemophilus influenzae has ushered in an era of unprecedented understanding into molecular aspects of intramembrane proteolysis by this fascinating class of protein. A combination of structural studies by X-ray crystallography, and biophysical and spectroscopic analyses, combined with traditional enzymatic and functional analysis has revealed fundamental aspects of rhomboid structure, substrate recognition and the catalytic mechanism. This review summarizes these remarkable advances by examining evidence for the proposed catalytic mechanism derived from inhibitor co-crystal structures, conflicting models of rhomboid-substrate interaction, and recent work on the structure and function of rhomboid cytosolic domains. In addition to exploring progress on aspects of rhomboid structure, areas for future research and unaddressed questions are emphasized and highlighted. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Regulation by proteolysis: energy-dependent proteases and their targets.

A number of critical regulatory proteins in both prokaryotic and eukaryotic cells are subject to rapid, energy-dependent proteolysis. Rapid degradation combined with control over biosynthesis provides a mechanism by which the availability of a protein can be limited both temporally and spatially. Highly unstable regulatory proteins are involved in numerous biological functions, particularly at the commitment steps in developmental pathways and in emergency responses. The proteases involved in energy-dependent proteolysis are large proteins with the ability to use ATP to scan for appropriate targets and degrade complete proteins in a processive manner. These cytoplasmic proteases are also able to degrade many abnormal proteins in the cell.     

高等植物中蛋白磷酸酶2C的结构与功能

蛋白质磷酸化／去磷酸化是生物信号级联传递的重要方式之一,主要通过生化性质互为对立的蛋白激酶和蛋白磷酸酶实现。蛋白磷酸酶2C(PP2C)是蛋白磷酸酶的一个分支,其生化性质、蛋白质组成与结构都和其他磷酸酶显著不同,但都在生物信号传递中扮演重要角色。高等植物中PP2C广泛参与脱落酸(ABA)的各种信号途径,包括ABA诱导的种子萌发／休眠、保卫细胞及离子通道调控和气孔关闭、逆境胁迫等。PP2C也多样地参与植物创伤反应、生长发育以及抗病性等各个途径。作为大多数信号途径的负调控因子,PP2C能直接与激酶结合,与其他调控蛋白结合,以及直接与DNA结合调控相关基因的表达。     

Intracellular proteolysis: signals of selective protein degradation

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases.     

Modulation of sensitivity and selectivity in plant signaling by proteasomal destabilization

The ubiquitin (Ub) system of intracellular protein degradation regulates the abundance of numerous proteins that control plant growth and development. Recent advances have begun to illustrate how environmental and endogenous signals affect plant responses through Ub-related proteolysis, the importance of combinatorial control in regulated protein destruction and how multiprotein complexes confer sensitivity and selectivity to ubiquitination. Further insight into the cell biology of Ub-chain assembly and proteasomal degradation, as well as into the relationship between proteolysis and other regulatory modifications, will be essential for understanding the mechanistic basis of the integration of diverse plant signals.     

Some Properties of Neutral Proteolytic System in Rabbit Skeletal Muscle

The properties of the neutral proteolytic activity concentrated in a fraction (F–1) separated from rabbit muscle homogenate were examined by measuring the effects of various reagents and metal ions, the time course of the proteolysis and Ca-stability. The obtained results have indicated that F–1 contains two types of neutral protease active on proteins, tentatively named Protease I and II, The former, which is activated by Ca²⁺ and Ca-labile, shows an explosive production of Cu-Folin phenol reagent positive materials at the early stage of incubation. The latter, which is Ca-stable, shows a large production of ninhydrin positive materials throughout the incubation time. The proteolysis by F–1 was similar to the autolysis of muscle homogenate in all the properties examined. Therefore, Proteases I and II were assumed to be main enzymes responsible for the muscle proteolysis at the neutral pH region. As there has been no factor denying their functioning in living muscle, it is probable that Proteases I and II take important parts in the muscle catabolism.     

AraPerox. A database of putative Arabidopsis proteins from plant peroxisomes

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.     

Intracellular protein degradation: basis of a self-regulating mechanism for the proteolysis of endogenous proteins

The intracellular basal proteolysis system, as distinct from the lysosomal system, is important in sustaining a high flux of proteins required for maintenance, growth and adaptability of cells. Its activity automatically fluctuates with changes in protein synthetic activity, but with a considerably slower response time, since the two processes are only indirectly or passively linked. Since as much as one-third of intracellular proteolysis in mammalian cells is directed as nascent proteins, the consequences are more fully discussed in relation to cell growth state. During rapid growth, cells have to accumulate more than double their original protein mass in order to achieve a 100% increase between divisions. The effects of reducing protein synthesis by inducing quiescence, serum step-down or cycloheximide treatment on intracellular proteolysis are considered, and the possibility that this leads to enhanced degradation of existing proteins has been explored. No substantial evidence was found to support this latter notion. The basal proteolysis system is seen as a constitutive, pervasive and broad-spectrumed collection of hydrolytic enzymes. It destroys proteins randomly, having no means of distinguishing young from old, aberrant from normal. The rate of demise of protein substrates depends on two factors, the ease of access of the hydrolytic enzymes to their peptide bonds, and the length of time that any species of protein remains at risk to this hydrolytic potential. While the former has long been recognized, the importance of the second factor in relation to the ability of proteins to become integrated in the living fabric of the cell is only beginning to be appreciated. The discussion also suggests elaborate regulatory mechanisms akin to those for protein synthesis would be unnecessary for protein degradation, especially if it can now be substantiated that substrate availability determines the turnover rates of proteins by a pervasive and relatively unlimited proteolytic system (Grisolía, 1964).     

Intracellular proteolysis: Signals of selective protein degradation

Selective proteolysis is one of the mechanisms for the maintenance of cell homeostasis via rapid degradation of defective polypeptides and certain short-lived regulatory proteins. In prokaryotic cells, high-molecular-mass oligomeric ATP-dependent proteases are responsible for selective protein degradation. In eukaryotes, most polypeptides are attacked by the multicatalytic 26S proteasome, and the degradation of the majority of substrates involves their preliminary modification with the protein ubiquitin. The proteins undergoing the selective proteolysis often contain specific degradation signals necessary for their recognition by the corresponding proteases. This article is dedicated to the 25th Anniversary of the journal Bioorganicheskaya Khimiya     

ATP-dependent Clp Proteases in Photosynthetic Organisms— A Cut Above the Rest!

Proteases are critical regulatory factors for many metabolic cellular processes as well as being vital for degrading proteins damaged during environmental stresses. Many of those responsible for targeted protein degradation require the hydrolysis of ATP, and one class that has attracted much attention recently are the Clp proteases. They are among the best characterized proteases to date, and were the first shown to rely on an ATPase regulatory subunit possessing molecular chaperone activity, which functions both within the proteolytic complex and independently. A range of Clp proteins has been identified from many different bacteria and eukaryotes, with by far the greatest number and diversity of forms in oxygenic photobionts such as cyanobacteria and higher plants. Functionally, Clp proteins have also evolved into one of the more critical proteolytic enzymes within photobionts, and it is now somewhat of a paradox that we currently know least about Clp protease functions in the photosynthetic organisms, where they have their most important roles. This discrepancy is now being addressed, with studies on Clp protein in cyanobacteria and, in an increasing number, in higher plants.     

Minor proteins and enzymes of the Drosophila eggshell matrix

The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.     

An essential protease involved in bacterial cell-cycle control.

Proteolytic inactivation of key regulatory proteins is essential in eukaryotic cell-cycle control. We have identified a protease in the eubacterium Caulobacter crescentus that is indispensable for viability and cell-cycle progression, indicating that proteolysis is also involved in controlling the bacterial cell cycle. Mutants of Caulobacter that lack the ATP-dependent serine protease ClpXP are arrested in the cell cycle before the initiation of chromosome replication and are blocked in the cell division process. ClpXP is composed of two types of polypeptides, the ClpX ATPase and the ClpP peptidase. Site-directed mutagenesis of the catalytically active serine residue of ClpP confirmed that the proteolytic activity of ClpXP is essential. Analysis of mutants lacking ClpX or ClpP revealed that both proteins are required in vivo for the cell-cycle-dependent degradation of the regulatory protein CtrA. CtrA is a member of the response regulator family of two-component signal transduction systems and controls multiple cell-cycle processes in Caulobacter. In particular, CtrA negatively controls DNA replication and our findings suggest that specific degradation of the CtrA protein by the ClpXP protease contributes to G1-to-S transition in this organism.