Cleavage of pyridoxal kinase into two structural domains: Kinetics of proteolysis monitored by emission anisotropy

Two sulfhydryl residues/dimer of pyridoxal kinase react with iodoacetamide fluoresceine (IAF) to yield catalytically active species. Limited chymotryptic digestion of IAF pyridoxal kinase resulted in the release of two fragments of 24 and 16 KDA. One of the fragments (16 KDA) is labeled with IAF. After complete tryptic digestion of IAF-pyridoxal kinase, only one peptide labeled with IAF was separated by reverse-phase HPLC and its amino acid sequence determined by automated Edman degradation. The kinetics of chymotryptic cleavage of IAF-pyridoxal kinase was monitored by steady-state emission anisotropy measurements. Analysis of the kinetic results revealed that the rate of proteolysis is significantly reduced by the substrate pyridoxal (0.2 mM). ATP (1 mM) does not influence the rate of proteolysis. The technique of emission anisotropy was also applied to monitor the effect of viscosity on the rate of proteolysis. A kinetic model is proposed to explain the mechanism of limited proteolysis. The model is based on the assumption that unfolding of the native conformation of the protein-substrate complex plays a dominant role in proteolysis.     

Proteolytic cleavage sites in smooth muscle myosin-light-chain kinase and their relation to structural and regulatory domains

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-II-1 and unc-II-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-II or unc-I domains or between the adjacent unc-II-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787-807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al. (1989) [Ikebe, M., Maruta, S. & Reardon, S. (1989) J. Biol. Chem. 264, 6967-6971] to be important in autoregulation of the myosin-light-chain kinase.     

Proteolytic cleavage of PDZD2 generates a secreted peptide containing two PDZ domains

PDZD2 (PDZ-domain-containing 2; also known as PAPIN, AIPC and PIN1) is a ubiquitously expressed multi-PDZ-domain protein. We have shown that PDZD2, which shows extensive homology to pro-interleukin-16 (pro-IL-16), is localized mainly to the endoplasmic reticulum (ER). Pro-IL-16 is cleaved in a caspase-3-dependent mechanism to generate the secreted cytokine IL-16. The abundant expression of PDZD2 in the ER, and its sequence similarity to pro-IL-16, suggests that similar post-translational processing of PDZD2 may occur. Indeed, western blotting and mass spectrometry analysis of conditioned medium from cells transfected with epitope-tagged PDZD2 show that there is secretion of a PDZD2 peptide of approximately 37 kDa (sPDZD2, for secreted PDZD2) that contains two PDZ domains. Expression of PDZD2 was detected in several tissues. Furthermore, sPDZD2 secretion is suppressed by the mutation of a sequence that shows similarity to caspase recognition motifs or by treatment with a caspase inhibitor. In summary, PDZD2 is the first reported multi-PDZ protein that is processed by proteolytic cleavage to generate a secreted peptide containing two PDZ domains.     

Proteolytic cleavage of Hansenula anomala flavocytochrome b2 into its two functional domains. Isolation of a highly active flavodehydrogenase and a cytochrome b2 core

In a previous work, we have described the tryptic cleavage of yeast flavocytochrome b2 into its two functional domains: a cytochrome b2 core and a flavodehydrogenase. The lactate dehydrogenase efficiency of the latter was, however, dramatically low, only about 1% that of intact flavocytochrome b2. Our present study concerns a new flavodehydrogenase derivative of Hansenula anomala flavocytochrome b2 which spontaneously dissociates from the cytochrome domain when the polypeptide bridge connecting them is cleaved by Staphylococcus aureus V8 protease I. This flavodehydrogenase was purified and some of its functional and structural properties were studied. It presents an exceptionally high lactate dehydrogenase activity, about 80% that of flavocytochrome b2. This result clearly demonstrates that the cytochrome domain is not necessary for the lactate dehydrogenase function and suggests an autonomous folding for both domains. Our results are discussed in terms of 'gene fusion'.     

Proteolytic cleavage of the catalytic subunit of DNA-dependent protein kinase during poliovirus infection

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK(CS). Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK(CS) is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK(CS) enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK(CS) is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK(CS) is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK(CS) is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK(CS) during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.     

Proteolytic cleavage during chemotherapy-induced apoptosis

Treatment with anticancer drugs sets into motion a morphologically and biochemically distinct type of cell death called apoptosis. Recent genetic and biochemical studies have suggested that proteases play a prominent role in the active phase of apoptotic cell death. Ongoing studies are aimed at identifying the proteases involved, the substrates that are cleaved, and the means by which the proteolytic process is regulated in nonapoptotic and apoptotic cells. The possibility that these findings will suggest new approaches to treating cancer and other diseases is discussed.     

Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells

Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.     

Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B(6), pyridoxal 5'-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B(6) is converted to PLP by PL kinase. PLP is the B(6) vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B(6), or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.     

Proteolytic cleavage and structural transformation: Their relationship in bacteriophage T4 capsid maturation

Giant T4 phage capsoids formed in canavanine-treated cultures infected by phage mutants in genes 21 and 17, respectively, differ with regard to cleavage of the major capsid protein, gp 23, and in the fine structure of their hexagonal surface lattices. Quantitative computer processing of electron micrographs shows that the significant differences in capsomer morphology amount to six symmetrically placed features present in the uncleaved hexamer but absent after cleavage. These features may be related with the N-terminal portions of gp 23 monomers excised by phage-specific proteolysis. Cleaved 17^? giants can be induced to undergo a further structural transformation (expansion). Structural characteristics of partially transformed giant particles give clues about the dynamics of the cleavage and expansion transformations. Both processes appear to be polar, initiating in one cap and propagating along the particle. The transition zone of partial cleavage is diffuse, whereas the transition between unexpanded and expanded areas is confined to a narrow band of some 20 nm width.     

Binding of a photoaffinity analogue of pyridoxal to pyridoxal kinase

The binding of pyridoxal analogues to the structural domains of pyridoxal kinase was studied by fluorescence spectroscopy and chromatographic techniques. Two fragments of 24 and 16 kDa, arising from limited proteolysis of the native enzyme, were separated by ion-exchange chromatography and used for binding studies with pyridoxal oxime. Fluorometric titrations yielded dissociation constants of 6 and 12.4 MicroM for pyridoxal oxime bound to the native enzyme and 24-kDa fragment, respectively. 4-(4-Azido-2-nitrophenyl)-pyridoxamine, a new photolabeling reagent, binds irreversibly to the kinase with concomitant loss of catalytic activity. The modified kinase (2.1 mol label/mol dimer) yields two fragments upon limited proteolysis with chymotrypsin. The two fragments were separated by reverse-phase HPLC and SDS/polyacrylamide gel electrophoresis. Radiolabeled ligand was detected only in the 24-kDa fragment. It is postulated that the pyridoxal binding site is located in the 24-kDa structural domain.     

Tat-mediated protein transduction of human brain pyridoxal kinase into PC12 cells

Pyridoxal kinase (PK) catalyses the phosphorylation of vitamin B6 to pyridoxal-5'-phosphate (PLP). A human brain PK gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-PK fusion protein. The expressed and purified Tat-PK fusion proteins transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously in culture media. Once inside the cells, the transduced Tat-PK proteins showed catalytic activity and are stable for 48 h. The intracellular concentration of PLP, which is known as a biologically active form of vitamin B6, was increased by pre-treatment of Tat-PK to the PC12 cells. Those results suggest that the transduction of Tat-PK fusion protein can be one of the ways to regulate the PLP level and to replenish this enzyme in the various neurological disorders related to vitamin B6.     

Proteolytic cleavage of Ii to p25

The 25,000-Da protein that is seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii was shown to be a C-terminal fragment of Ii. [35S]Methionine pulse-chase labeling of polyclonally activated B lymphocytes or lymphoblastoid cell lines demonstrated maximal appearance of p25 in Percoll-separated endosomal fractions at 20- to 40-min chase times (studies in progress). This finding was consistent with the view that proteolysis of Ii to p25 and its release might catalyze the binding of digested foreign peptides to class II molecules and/or govern release of such charged complexes to traffic to the cell surface. We examined the structural relationship of p25 to Ii and the basis for cleavage of a relatively restricted site just external to the transmembranal segment. [35S]Methionine-labeled Ii and associated molecules were immunoprecipitated with a mAb to native Ii and then denatured, resolubilized, and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-terminal peptides of Ii (183 to 193 and 192 to 211), but not antibodies to an N-terminal peptide (12 to 28), did immunoprecipitate p25. The three antibodies to C-terminal and N-terminal peptides all immunoprecipitated denatured Ii proteins. The mAb to Ii immunoprecipitated [35S]methionine-labeled p25 but not [35S]cysteine-labeled p25. This finding was consistent with loss of a portion of Ii containing the only cysteine in Ii, Cys28. Digestion of class II MHC Ag-Ii complexes with various proteases yielded proteins migrating at and near p25 in two-dimensional electrophoretic gels. Upon increasing the duration of protease digestion, the 25,000-Da fragments were relatively resistant to further digestion. This observation was consistent with the presence of secondary structures (domains) leaving a relatively protease-sensitive (Ig hinge-like) region in Ii near its insertion into the membrane.     

Proteolytic cleavage of Beclin 1 exacerbates neurodegeneration

Background

Neuronal cell loss contributes to the pathology of acute and chronic neurodegenerative diseases, including Alzheimer’s disease (AD). It remains crucial to identify molecular mechanisms sensitizing neurons to various insults and cell death. To date, the multifunctional, autophagy-related protein Beclin 1 has been shown to be both necessary and sufficient for neuronal integrity in neurodegenerative models associated with protein aggregation. Interestingly, besides its role in cellular homeostasis, Beclin 1 has also been ascribed a role in apoptosis. This makes it critical to elucidate whether Beclin 1 regulates neuronal death and survival across neurodegenerative conditions independent of protein clearance. Here, we provide experimental evidence for a direct functional link between proteolytic cleavage of Beclin 1 and apoptotic neuronal cell loss in two independent models of neurodegeneration in vivo.

Methods

Proteolytic cleavage of Beclin 1 was characterized in lysates of human AD brain samples. We developed viral tools allowing for the selective neuronal expression of the various Beclin 1 forms, including Beclin 1 cleavage products as well as a cleavage-resistant form. The effect of these Beclin 1 forms on survival and integrity of neurons was examined in models of acute and chronic neurodegeneration in vitro and in vivo. Markers of neuronal integrity, neurodegeneration and inflammation were further assessed in a Kainic acid-based mouse model of acute excitotoxic neurodegeneration and in a hAPP-transgenic mouse model of AD following perturbation of Beclin 1 in the susceptible CA1 region of the hippocampus.

Results

We find a significant increase in caspase-mediated Beclin 1 cleavage fragments in brain lysates of human AD patients and mimic this phenotype in vivo using both an excitotoxic and hAPP-transgenic mouse model of neurodegeneration. Surprisingly, overexpression of the C-terminal cleavage-fragment exacerbated neurodegeneration in two distinct models of degeneration. Local inhibition of caspase activity ameliorated neurodegeneration after excitotoxic insult and prevented Beclin 1 cleavage. Furthermore, overexpression of a cleavage-resistant form of Beclin 1 in hippocampal neurons conferred neuroprotection against excitotoxic and Amyloid beta-associated insults in vivo.

Conclusions

Together, these findings indicate that the cleavage state of Beclin 1 determines its functional involvement in both neurodegeneration and neuroprotection. Hence, manipulating the cleavage state of Beclin 1 may represent a therapeutic strategy for preventing neuronal cell loss across multiple forms of neurodegeneration.

     

Activation of pyridoxal kinase by metallothionein

Brain pyridoxal kinase, which uses ATP complexed to either Zn(II) or Co(II) as substrates, displays high catalytic activity in the presence of Zn-thionein and Co-thionein. Several steps intervene in the process of pyridoxal kinase activation, i.e., binding of Zn ions to ATP and interaction between Zn-ATP and the enzyme. Equilibrium binding studies show that ATP mediates the release of Zn ions from the metal-thiolate clusters of the thioneins, whereas spectroscopic measurements conducted on Co-thionein reveal that the absorption transitions corresponding to the metal-thiolate of the protein are perturbed by ATP. The binding Zn-ATP to the kinase proceeds with a delta G = -6.3 kcal/mol as demonstrated by fluorometric titrations. Direct interaction between the kinase and derivatized-metallothionein could not be detected by emission anisotropy measurements, indicating that juxtaposition of the proteins does not influence the exchange of metal ions. Since the concentration of free Zn in several mammalian tissues is lower than 1 nM, it is postulated that under in vivo conditions the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase.     

Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes

A detailed comparison of the structures of aspartate aminotransferase, alanine race-mase, the beta subunit of tryptophan synthase, D-amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)-binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent alpha-beta-alpha-beta supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous alpha-beta-alpha-beta structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements.     

Interrelationships between pyridoxal phosphate and pyridoxal kinase in rabbit brain

—An inverse relationship was demonstrable between the concentration of pyridoxal phosphate and the activity of pyridoxal kinase in rabbit brain. The administration of pyridoxine elevated the concentration of pyridoxal phosphate and decreased the activity of pyridoxal kinase. Conversely, the administration of deoxypyridoxine decreased the concentration of pyridoxal phosphate and increased the activity of pyridoxal kinase. The increase in the activity of pyridoxal kinase by deoxypyridoxine was blocked by actinomycin D or puromycin. These results were interpreted to indicate that the tissue availability of pyridoxal phosphate regulated the activity of pyridoxal kinase.