Detection of modification by ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins (Ubls) are conjugated to many target proteins either as monomeric units or as polymeric chains. There are at least 12 members of the ubiquitin family in the human genome and their conjugation dramatically alters the properties of the modified protein. The presence of highly active proteases that specifically deconjugate Ubls often means that, in the cell, the steady state level of modified protein is low. Detection of protein species modified by Ubls can therefore represent a significant challenge. Here, we describe methods that have been developed to allow detection of Ubl modified proteins both in vivo and in vitro.     

Posttranslational modification of proteins by isoprenoids in mammalian cells

Isoprenylation is a posttranslational modification that involves the formation of thioether bonds between cysteine and isoprenyl groups derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. Numerous isoprenylated proteins have been detected in mammalian cells. Those identified include K-, N-, and H-p21ras, ras-related GTP-binding proteins such as G25K (Gp), nuclear lamin B and prelamin A, and the gamma subunits of heterotrimeric G proteins. The modified cysteine is located in the fourth position from the carboxyl terminus in every protein where this has been studied. For p21ras, the last three amino acids are subsequently removed and the exposed cysteine is carboxylmethylated. Similar processing events may occur in lamin B and G protein gamma subunits, but the proteolytic cleavage in prelamin A occurs upstream from the modified cysteine. Lamin B and p21ras are modified by C15 farnesyl groups, whereas other proteins such as the G protein gamma subunits are modified by C20 geranylgeranyl chains. Separate enzymes may catalyze these modifications. The structural features that govern the ability of particular proteins to serve as substrates for isoprenylation by C15 or C20 groups are not completely defined, but studies of the p21ras modification using purified farnesyl:protein transferase suggest that the sequence of the carboxyl-terminal tetrapeptide is important. Isoprenylation plays a critical role in promoting the association of p21ras and the lamins with the cell membrane and nuclear envelope, respectively. Future studies of the role of isoprenylation in the localization and function of ras-related GTP-binding proteins and signal-transducing G proteins should provide valuable new insight into the link between isoprenoid biosynthesis and cell growth.     

Posttranslational modification of autophagy-related proteins in macroautophagy

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.     

Posttranslational modification of therapeutic proteins in plants

Plants have emerged as an alternative to current systems for the production of therapeutic proteins. The advantages of plants for the low-cost and large-scale production of safe and biologically active mammalian proteins have been documented recently. A major advantage of transgenic plants over production systems that are based on yeast or Escherichia coli is their ability to perform most of the posttranslational modifications (PTMs) that are required for the bioactivity and pharmacokinetics of recombinant therapeutic proteins. Furthermore, recent advances in the control of PTMs in transgenic plants have made it possible for plants to perform, at least to some extent, human-like modifications of recombinant proteins. Hence, plants have become a suitable alternative to animal cell factories for the production of therapeutic proteins.     

Posttranslational modification of autophagy-related proteins in macroautophagy

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases.     

Toward minimalist models of larger proteins: a ubiquitin-like protein

Our recently developed off-lattice bead model capable of simulating protein structures with mixed alpha/beta content has been extended to model the folding of a ubiquitin-like protein and provides a means for examining the more complex kinetics involved in the folding of larger proteins. Using trajectories generated from constant-temperature Langevin dynamics simulations and sampling with the multiple multi-histogram method over five-order parameters, we are able to characterize the free energy landscape for folding and find evidence for folding through compact intermediates. Our model reproduces the observation that the C-terminus loop structure in ubiquitin is the last to fold in the folding process and most likely plays a spectator role in the folding kinetics. The possibility of a productive metastable intermediate along the folding pathway consisting of collapsed states with no secondary structure, and of intermediates or transition structures involving secondary structural elements occurring early in the sequence, is also supported by our model. The kinetics of folding remain multi-exponential below the folding temperature, with glass-like kinetics appearing at T/T(f) approximately 0.86. This new physicochemical model, designed to be predictive, helps validate the value of modeling protein folding at this level of detail for genomic-scale studies, and motivates further studies of other protein topologies and the impact of more complex energy functions, such as the addition of solvation forces.     

The ubiquitin-like protein HUB1 forms SDS-resistant complexes with cellular proteins in the absence of ATP

Ubiquitin and ubiquitin-like modifiers (UBLs) form covalent complexes with other proteins by isopeptide formation between their carboxyl (C)-termini and -amino groups of lysine residues of acceptor proteins. A hallmark of UBLs is a protruding C-terminal tail with a terminal glycine residue, which is required for ATP-dependent conjugation. Recently, the highly conserved protein HUB1 (homologous to ubiquitin 1) has been reported to function as a UBL following C-terminal processing. HUB1 exhibits sequence similarity with ubiquitin but lacks a C-terminal tail bearing a glycine residue. Here we show that HUB1 can form SDS-resistant complexes with cellular proteins, but provide evidence that these adducts are not formed through covalent C-terminal conjugation of HUB1 to substrates. The adducts are still formed when the C-terminus of HUB1 was altered by epitope tagging, amino-acid exchange or deletion, or when cells were depleted of ATP. We propose that HUB1 may act as a novel protein modulator through the formation of tight, possibly noncovalent interactions with target proteins.     

The cellular protein level of parkin is regulated by its ubiquitin-like domain

Parkin is a ubiquitin-protein isopeptide ligase (E3) involved in ubiquitin/proteasome-mediated protein degradation. Mutations in the parkin gene cause a loss-of-function and/or alter protein levels of parkin. As a result, the toxic build-up of parkin substrates is thought to lead to autosomal recessive juvenile Parkinsonism. To identify a role for the ubiquitin-like domain (ULD) of parkin, we created a number of hemagglutinin (HA)-tagged parkin constructs using mutational and structural information. Western blotting and immunocytochemistry showed a much stronger expression level for HA-parkin residues 77-465 (without ULD) than HA-parkin full-length (with ULD). The deletion of ULD in Drosophila parkin also caused a sharp increase in expression of the truncated form, suggesting that the function of the ULD of parkin is conserved across species. By progressive deletion analysis of parkin ULD, we found that residues 1-6 of human parkin play a crucial role in controlling the expression levels of this gene. HA-parkin residues 77-465 showed ubiquitination in vivo, demonstrating that the ULD is not critical for parkin auto-ubiquitination; ubiquitination seemed to cluster on the central domain of parkin (residues 77-313). These effects were specific for the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or vector-dependent. Taken together, these data suggest that the 76 most NH(2)-terminal residues (ULD) dramatically regulate the protein levels of parkin.     

Posttranslational processing of proteins in vacuoles and protein bodies is inhibited by monensin

A number of proteins that accumulate in vacuoles and protein bodies undergo posttranslational processing at these accumulation sites. These processing steps include proteolytic cleavage (e.g. pea lectin, soybean glycinin, and rice lectin) and the removal of some sugar residues from oligosaccharide side-chains (e.g. bean phytohemagglutinin). Treatment of immature rice embryos with the sodium ionophore monensin slows down the proteolytic processing of the rice lectin precursor (M_r 23,000) to mature rice lectin (M_r 10,000 and 8,000). Treatment of developing bean cotyledons with monensin slows down the removal of peripheral N-acetylglucosamine residues from the oligosaccharide side-chains of phytohemagglutinin. The results are consistent with the interpretation that these processing steps, which occur in vacuoles or protein bodies, are carried out by enzymes with an acidic pH optimum, and that monensin slows down processing by alkalinization of the vacuoles or protein bodies.     

Different roles for two ubiquitin-like domains of ISG15 in protein modification

ISG15 (interferon-stimulated gene 15) is a novel ubiquitin-like (UbL) modifier with two UbL domains in its architecture. We investigated different roles for the two UbL domains in protein modification by ISG15 (ISGylation) and the impact of Influenza B virus NS1 protein (NS1B) on regulation of the pathway. The results show that, although the C-terminal domain is sufficient to link ISG15 to UBE1L and UbcH8, the N-terminal domain is dispensable in the activation and transthiolation steps but required for efficient E3-mediated transfer of ISG15 from UbcH8 to its substrates. NS1B specifically binds to the N-terminal domain of ISG15 but does not affect ISG15 linkage via a thioester bond to its activating and conjugating enzymes. However, it does inhibit the formation of cellular ISG15 conjugates upon interferon treatment. We propose that the N-terminal UbL domain of ISG15 mainly functions in the ligation step and NS1B inhibits ISGylation by competing with E3 ligases for binding to the N-terminal domain.     

Posttranslational modification of MDM2

The functions of the MDM2 protein, in particular its E3 ubiquitin ligase activity and its ability to interact with a number of cellular proteins intimately involved in growth regulation, are modulated by sumoylation and multisite phosphorylation. These posttranslational mechanisms not only regulate the intrinsic activity of MDM2 in response to cellular stresses, but also govern its subcellular localization, differentiate between MDM2-mediated ubiquitination of p53 and autoubiquitination, integrate the stress response with mechanisms that mediate cell survival, and modulate the interaction of MDM2 with cellular and viral proteins. In this review, we summarize our current knowledge of the role of posttranslational modifications of MDM2 and their functional relevance.     

Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification

Products of ras genes are synthesized as precursors in the cytosol and transported to the plasma membrane by a process which involves posttraslational modification by fatty acid. In this paper, we present evidence for the occurrence in the cytosol of an intermediate modification of ras proteins prior to the fatty acid acylation. The modification is detected by a slight shift in the mobility of the protein on SDS polyacrylamide gel. The fatty acid acylation does not contribute to this mobility shift. This modification is affected by the dprl mutation which has recently been shown to affect the processing of yeast RAS proteins. To further characterize the nature of the modification event, we have cloned DPR1 gene from the DNA of Saccharomyces cerevisiae. The gene is actively transcribed in yeast cells producing mRNA of approximately 1.6 kb. Genes related to the DRP1 appear to be present in a distantly related yeast, Schizosaccharomyces pombe as well as in guinea pig and human cells.     

Regulation of translesion DNA synthesis: Posttranslational modification of lysine residues in key proteins

Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo.     

Posttranslational modification of serine to formylglycine in bacterial sulfatases. Recognition of the modification motif by the iron-sulfur protein AtsB

Calpha-formylglycine is the catalytic residue of sulfatases. Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. The modifying enzymes are unknown. AtsB, an iron-sulfur protein, is strictly required for modification of Ser(72) in the periplasmic sulfatase AtsA of Klebsiella pneumoniae. Here we show (i) that AtsB is a cytosolic protein acting on newly synthesized serine-type sulfatases, (ii) that AtsB-mediated FGly formation is dependent on AtsA's signal peptide, and (iii) that the cytosolic cysteine-type sulfatase of Pseudomonas aeruginosa can be converted into a substrate of AtsB if the cysteine is substituted by serine and a signal peptide is added. Thus, formylglycine formation in serine-type sulfatases depends both on AtsB and on the presence of a signal peptide, and AtsB can act on sulfatases of other species. AtsB physically interacts with AtsA in a Ser(72)-dependent manner, as shown in yeast two-hybrid and GST pull-down experiments. This strongly suggests that AtsB is the serine-modifying enzyme and that AtsB relies on a cytosolic function of the sulfatase's signal peptide.     

Posttranslational modification of the 20S proteasomal proteins of the archaeon Haloferax volcanii

20S proteasomes are large, multicatalytic proteases that play an important role in intracellular protein degradation. The barrel-like architecture of 20S proteasomes, formed by the stacking of four heptameric protein rings, is highly conserved from archaea to eukaryotes. The outer two rings are composed of alpha-type subunits, and the inner two rings are composed of beta-type subunits. The halophilic archaeon Haloferax volcanii synthesizes two different alpha-type proteins, alpha1 and alpha2, and one beta-type protein that assemble into at least two 20S proteasome subtypes. In this study, we demonstrate that all three of these 20S proteasomal proteins (alpha1, alpha2, and beta) are modified either post- or cotranslationally. Using electrospray ionization quadrupole time-of-flight mass spectrometry, a phosphorylation site of the beta subunit was identified at Ser129 of the deduced protein sequence. In addition, alpha1 and alpha2 contained N-terminal acetyl groups. These findings represent the first evidence of acetylation and phosphorylation of archaeal proteasomes and are one of the limited examples of post- and/or cotranslational modification of proteins in this unusual group of organisms.     

Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase

We demonstrate the ability of Pseudomonas putida KT2440, Pseudomonas syringae pv. tomato DC3000 and Pseudomonas stutzeri DSM10701 to posttranslationally activate carrier protein (CP) domains of various polyketide synthases, nonribosomal peptide synthetases, and fatty acid synthase by their intrinsic phosphopantetheinyl transferase. The apo-form is modified to the holo-form of the CP by attaching a phosphopantetheine moiety from coenzymeA to a conserved serine residue. The coding regions of the respective domains were cloned in order to generate C-terminal fusions with intein-chitin. The constructs were subcloned into a broad host range vector and transferred into the three pseudomonad hosts. The resulting recombinant pseudomonad strains were cultivated and each fusion protein was purified by affinity chromatography. Each purified CP was analysed using MALDI/TOF for the expected mass increase. Of the seven CPs tested, six could be purified from P. putida, which was chosen as the general host strain. Out of the six domains, five were completely activated, whereas only 5% of the protein of the sixth domain was in holo-form. Four domains were also expressed in the other hosts.     

Posttranslational modification of ornithine decarboxylase by its product putrescine

Putrescine, spermidine, and spermine, as well as other primary amine substances, when added exogenously to growth-stimulated systems, inhibit ornithine decarboxylase (ODC) activity in a dose- and time-dependent manner. Evidence is presented to support a direct posttranslational modification of ODC by transglutaminase-mediated putrescine incorporation. Purified ODC serves as an acceptor protein for putrescine in the presence of transglutaminase purified from guinea pig liver. The transamidation of putrescine to ODC results in a linear decrease in activity. The Km for putrescine is 0.4 mM and the Ki for putrescine inhibition of ODC activity by transglutaminase is 0.4 mM. The kinetics are identical to those reported for physiological systems. In regenerating rat liver, protein conjugated putrescine parallels increased transglutaminase activity and the rapid disappearance of ODC activity at 8 h. These data strongly suggest that posttranslational modification of ODC by putrescine may be an important regulatory step in the trophic cascade.