Granzyme M: characterization with sites of post-translational modification and specific sites of interaction with substrates and inhibitors

Granzymes kill cells in a variety of ways. They induce mitochondrial dysfunction through caspase dependent and caspase-independent pathways and destroy DNA and the integrity of the nucleus. For gaining a better understanding of the molecular function of granzyme M and its NK cell specificity, structural characterization of this enzyme by molecular modeling as well as its detailed comparison with other granzymes is presented in this study. The study includes mode of action of granzyme M using cationic binding sites, substrate specificity, post-translational structural modification and its functional relationship and interaction of the enzyme with inhibitor in an attempt to explore how the activity of human granzyme M is controlled under physiological conditions. It is concluded from the present study that the post-translational modification, including Oglycosylation of serine, phosphorylation of serine and threonine and myristoylation of glycine, play an important role in the interaction of enzyme with the cell surface membrane and regulate protein trafficking and stability. Phosphorylated serine and threonine also plays a role in tumor elimination, viral clearance and tissue repair. In Gzm M there are cationic sites, cs1 and cs2 that may participate in binding of Gzm M to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm. Gzm M shows apoptotic activity both by caspase dependent and independent pathways. Modeling of inhibitors bound to the granzyme active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft.     

Glycosylation is a novel TGFβ1-independent post-translational modification of Smad2

Smad2 is a crucial component of intracellular signaling by transforming growth factor-β (TGFβ). Here we describe that Smad2 is glycosylated, which is a novel for Smad2 post-translational modification. We showed that the Smad2 glycosylation was inhibited upon treatment of cells with 17β-estradiol, and was enhanced in cells in a dense culture as compared to cells in a sparse culture. The Smad2 glycosylation was not dependent on the C-terminal phosphorylation of Smad2, and was not affected by TGFβ1 treatment of the cells. Smad2 was glycosylated at multiple sites, and one of the predicted sites is Serine110. Thus, Smad2 is glycosylated, and this post-translational modification was modulated by 17β-estradiol but not by TGFβ1.     

The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range

The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants.     

Adenylylation: renaissance of a forgotten post-translational modification

The stable post-translational modification of proteins by adenylylation or uridylylation was discovered more than four decades ago as a mechanism to regulate the activity of enzymes. Although many other processes involving the covalent transfer of an AMP residue to an amino acid side chain have been identified since then, these are transient adenylylation events that essentially use the free energy of ATP hydrolysis to activate specific processes. Recently, new examples of stable adenylylation of small GTPases involved in signal transduction and regulation of cellular events were discovered, which appear to modulate downstream processes such as cytoskeletal rearrangement and vesicular trafficking. We present a survey of the historical and modern phases of research in this area, focusing on the common and differing aspects of protein adenylylation.     

Monitoring post-translational modification of proteins with allosteric ribozymes

An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.     

Potent pro-apoptotic combination therapy is highly effective in a broad range of cancers

Primary or acquired therapy resistance is a major obstacle to the effective treatment of cancer. Resistance to apoptosis has long been thought to contribute to therapy resistance. We show here that recombinant TRAIL and CDK9 inhibition cooperate in killing cells derived from a broad range of cancers, importantly without inducing detectable adverse events. Remarkably, the combination of TRAIL with CDK9 inhibition was also highly effective on cancers resistant to both, standard-of-care chemotherapy and various targeted therapeutic approaches. Dynamic BH3 profiling revealed that, mechanistically, combining TRAIL with CDK9 inhibition induced a drastic increase in the mitochondrial priming of cancer cells. Intriguingly, this increase occurred irrespective of whether the cancer cells were sensitive or resistant to chemo- or targeted therapy. We conclude that this pro-apoptotic combination therapy has the potential to serve as a highly effective new treatment option for a variety of different cancers. Notably, this includes cancers that are resistant to currently available treatment modalities.Subject terms: Cancer models, Cell biology, Preclinical research     

Oxidation as a post-translational modification that regulates autophagy

The toxicity associated with accumulation of reactive oxygen species (ROS) has led to the evolution of various defense strategies to overcome oxidative stress, including autophagy. This pathway is involved in the removal and degradation of damaged mitochondria and oxidized proteins. At low levels, however, ROS act as signal transducers in various intracellular pathways. In a recent study we described the role of ROS as signaling molecules in starvation-induced autophagy. We showed that starvation stimulates formation of ROS, specifically H(2)O(2), in the mitochondria. Furthermore, we identified the cysteine protease HsAtg4 as a direct target for oxidation by H(2)O(2), and specified a cysteine residue located near the HsAtg4 catalytic site as critical for this regulation. Here we focus on Atg4, the target of regulation, and discuss possible mechanisms for the regulation of this enzyme in the autophagic process.     

Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site

The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat alpha 1(I) gelatin, beta-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the alpha 1(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of lysine succinylation as a new post-translational modification

Of the 20 ribosomally coded amino acid residues, lysine is the most frequently post-translationally modified, which has important functional and regulatory consequences. Here we report the identification and verification of a previously unreported form of protein post-translational modification (PTM): lysine succinylation. The succinyllysine residue was initially identified by mass spectrometry and protein sequence alignment. The identified succinyllysine peptides derived from in vivo proteins were verified by western blot analysis, in vivo labeling with isotopic succinate, MS/MS and HPLC coelution of their synthetic counterparts. We further show that lysine succinylation is evolutionarily conserved and that this PTM responds to different physiological conditions. Our study also implies that succinyl-CoA might be a cofactor for lysine succinylation. Given the apparent high abundance of lysine succinylation and the significant structural changes induced by this PTM, it is expected that lysine succinylation has important cellular functions.     

Tobacco mitochondrial small heat shock protein NtHSP24.6 adopts a dimeric configuration and has a broad range of substrates

There is a broad range of different small heat shock proteins (sHSPs) that have diverse structural and functional characteristics. To better understand the functional role of mitochondrial sHSP, NtHSP24.6 was expressed in Escherichia coli with a hexahistidine tag and purified. The protein was analyzed by non-denaturing PAGE, chemical cross-linking and size exclusion chromatography and the H6NtHSP24.6 protein was found to form a dimer in solution. The in vitro functional analysis of H6NtHSP24.6 using firefly luciferase and citrate synthase demonstrated that this protein displays typical molecular chaperone activity. When cell lysates of E. coli were heated after the addition of H6NtHSP24.6, a broad range of proteins from 10 to 160 kD in size remained in the soluble state. These results suggest that NtHSP24.6 forms a dimer and can function as a molecular chaperone to protect a diverse range of proteins from thermal aggregation.     

Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates

Aims:  Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest.
Methods and Results:  Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25°C, indicating the secretion of active enzyme by the fungus, and one-third produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15°C and some at 39°C.
Conclusions:  Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates.
Significance and Impact of the Study:  To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities.     

Palmitoylation is a post-translational modification of Alix regulating the membrane organization of exosome-like small extracellular vesicles

Background

Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions.

Poly ADP-ribosylation of proteins. Processivity of a post-translational modification

The nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) participates in DNA excision repair by post-translational selfmodification ("automodification") and the modification of other chromatin proteins ("heteromodification") with ADP-ribose polymers. We have studied the molecular mechanism of these reactions in a reconstituted in vitro system. After activation by DNA, poly(ADP-ribose) polymerase produces polymers with a distinct size pattern. These polymers are attached to a small subfraction of enzyme molecules. As the reaction progresses, more enzyme molecules are recruited for modification with an identical polymer size pattern. Likewise, the auto- and heteromodification reaction in nucleosomal core particles involves the consecutive addition of a highly conserved polymer size pattern to the acceptor proteins. Thus, a highly conserved polymer size pattern may constitute the molecular signal priming chromatin proteins for a role in DNA excision repair in vivo. The priming reaction is processive.     

Synthesis of probes with broad pH range fluorescence

Reaction of a rhodamine 2'-ester with an excess of alkyldiamines provides amino-functionalized rhodamine spirolactams, which when subsequently conjugated with carboxyfluorescein, provides probes which are fluorescent at acidic, neutral, and basic pH ranges.