Structure of the HIV-1 integrase catalytic domain complexed with an inhibitor: A platform for antiviral drug design

HIV integrase, the enzyme that inserts the viral DNA into the host chromosome, has no mammalian counterpart, making it an attractive target for antiviral drug design. As one of the three enzymes produced by HIV, it can be expected that inhibitors of this enzyme will complement the therapeutic use of HIV protease and reverse transcriptase inhibitors. We have determined the structure of a complex of the HIV-1 integrase core domain with a novel inhibitor, 5ClTEP, 1-(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone, to 2.1-Å resolution. The inhibitor binds centrally in the active site of the integrase and makes a number of close contacts with the protein. Only minor changes in the protein accompany inhibitor binding. This inhibitor complex will provide a platform for structure-based design of an additional class of inhibitors for antiviral therapy.

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

Tsetse thrombin inhibitor: Bloodmeal-induced expression of an anticoagulant in salivary glands and gut tissue of Glossina morsitans morsitans

The tsetse thrombin inhibitor, a potent and specific low molecular mass (3,530 Da) anticoagulant peptide, was purified previously from salivary gland extracts of Glossina morsitans morsitans (Diptera: Glossinidae). A 303-bp coding sequence corresponding to the inhibitor has now been isolated from a tsetse salivary gland cDNA library by using degenerate oligonucleotide probes. The full-length cDNA contains a 26-bp untranslated segment at its 5′ end, followed by a 63-bp sequence corresponding to a putative secretory signal peptide. A 96-bp segment codes for the mature tsetse thrombin inhibitor, whose predicted molecular weight matches that of the purified native protein. Based on its lack of homology to any previously described family of molecules, the tsetse thrombin inhibitor appears to represent a unique class of naturally occurring protease inhibitors. Recombinant tsetse thrombin inhibitor expressed in Escherichia coli and the chemically synthesized peptide are both substantially less active than the purified native protein, suggesting that posttranslational modification(s) may be necessary for optimal inhibitory activity. The tsetse thrombin inhibitor gene, which is present as a single copy in the tsetse genome, is expressed at high levels in salivary glands and midguts of adult tsetse flies, suggesting a possible role for the anticoagulant in both feeding and processing of the bloodmeal.

Unexpected crucial role of residue 225 in serine proteases

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically influences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation.

Substrate sequestration by a proteolytically inactive Lon mutant

Lon protein of Escherichia coli is an ATP-dependent protease responsible for the rapid turnover of both abnormal and naturally unstable proteins, including SulA, a cell division inhibitor made after DNA damage, and RcsA, a positive regulator of transcription. Lon is a multimer of identical 94-kDa subunits, each containing a consensus ATPase motif and a serine active site. We found that overexpressing Lon, which is mutated for the serine active site (LonS679A) and is therefore devoid of proteolytic activity, unexpectedly led to complementation of the UV sensitivity and capsule overproduction of a lon deletion mutant. SulA was not degraded by LonS679A, but rather was completely protected by the Lon mutant from degradation by other cellular proteases. We interpret these results to mean that the mutant LonS679A binds but does not degrade Lon substrates, resulting in sequestration of the substrate proteins and interference with their activities, resulting in apparent complementation. Lon that carried a mutation in the consensus ATPase site, either with or without the active site serine, was no longer able to complement a Δlon mutant. These in vivo results suggest that the pathway of degradation by Lon couples ATP-dependent unfolding with movement of the substrate into protected chambers within Lon, where it is held until degradation proceeds. In the absence of degradation the substrate remains sequestered. Comparison of our results with those from a number of other systems suggest that proteins related to the regulatory portions of energy-dependent proteases act as energy-dependent sequestration proteins.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

The cyclin-dependent kinase inhibitor p27Kip1 induces N-terminal proteolytic cleavage of cyclin A

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27Kip1 (p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G1. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15Ink4b, p16Ink4a, p21Cip1, and p57Kip2) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (pTwenty-seven- activated protease).

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Activation of a protease cascade involved in patterning the Drosophila embryo

Dorsoventral patterning of the Drosophila embryo is initiated by a ventralizing signal. Production of this signal requires the serine proteases Gastrulation Defective (GD), Snake, and Easter, which genetic studies suggest act sequentially in a cascade that is activated locally in response to a ventral cue provided by the pipe gene. Here, we demonstrate biochemically that GD activates Snake, which in turn activates Easter. We also provide evidence that GD zymogen cleavage is important for triggering this cascade but is not spatially localized by pipe. Our results suggest that a broadly, rather than locally, activated protease cascade produces the ventralizing signal, so a distinct downstream step in this cascade must be spatially regulated to restrict signaling to the ventral side of the embryo.

A new strategy to decrease N-methyl-d-aspartate (NMDA) receptor coactivation: Inhibition of d-serine synthesis by converting serine racemase into an eliminase

Serine racemase is a brain-enriched enzyme that synthesizes d-serine, an endogenous modulator of the glycine site of N-methyl-d-aspartate (NMDA) receptors. We now report that serine racemase catalyzes an elimination reaction toward a nonphysiological substrate that provides a powerful tool to study its neurobiological role and will be useful to develop selective enzyme inhibitors. Serine racemase catalyzes robust elimination of l-serine O-sulfate that is 500 times faster than the physiological racemization reaction, generating sulfate, ammonia, and pyruvate. This reaction provides the most simple and sensitive assay to detect the enzyme activity so far. We establish stable cell lines expressing serine racemase and show that serine racemase can also be converted into a powerful eliminase in cultured cells, while the racemization of l-serine is inhibited. Likewise, l-serine O-sulfate inhibits the synthesis of d-serine in primary astrocyte cultures. We conclude that the synthetic compound l-serine O-sulfate is a better substrate than l-serine as well as an inhibitor of d-serine synthesis. Inhibition of serine racemase provides a new strategy to selectively decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.

BRS1, a serine carboxypeptidase, regulates BRI1 signaling in Arabidopsis thaliana

Brassinosteroid-insensitive 1 (BRI1) of Arabidopsis thaliana encodes a cell surface receptor for brassinosteroids. Mutations in BRI1 severely affect plant growth and development. Activation tagging of a weak bri1 allele (bri1-5) resulted in the identification of a new locus, brs1-1D. BRS1 is predicted to encode a secreted carboxypeptidase. Whereas a brs1 loss-of-function allele has no obvious mutant phenotype, overexpression of BRS1 can suppress bri1 extracellular domain mutants. Genetic analyses showed that brassinosteroids and a functional BRI1 protein kinase domain are required for suppression. In addition, overexpressed BRS1 missense mutants, predicted to abolish BRS1 protease activity, failed to suppress bri1-5. Finally, the effects of BRS1 are selective: overexpression in either wild-type or two other receptor kinase mutants resulted in no phenotypic alterations. These results strongly suggest that BRS1 processes a protein involved in an early event in the BRI1 signaling.

Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors

The three-dimensional structures of the inactive protein precursors (zymogens) of the serine, cysteine, aspartic, and metalloprotease classes of proteolytic enzymes are known. Comparisons of these structures with those of the mature, active proteases reveal that, in general, the preformed, active conformations of the residues involved in catalysis are rendered sterically inaccessible to substrates by the residues of the zymogens’ N-terminal extensions or prosegments. The prosegments interact in nonsubstrate-like fashions with the residues of the active sites in most of the cases. The gastric aspartic proteases have a well-characterized zymogen conversion pathway. Structures of human progastricsin, the inactive intermediate 2, and active human pepsin are known and have been used to define the conversion pathway. The structure of the zymogen precursor of plasmepsin II, the malarial aspartic protease, shows a new twist on the mode of inactivation used by the gastric zymogens. The prosegment of proplasmepsin disrupts the active conformation of the two catalytic aspartic acid residues by inducing a major reorientation of the two domains of the mature protease. The picornaviral 2A and 3C proteases have a chymotrypsin-like tertiary structure but with a cysteine nucleophile. These enzymes cleave themselves from the viral polyprotein in cis (intramolecular cleavage) and carry out trans cleavages of other scissile peptides important for the virus life cycle. Although the structure of the precursor viral polyprotein is unknown, it probably resembles the organization of the proenzymes of the bacterial serine proteases, subtilisin, and α-lytic protease. Cleavage of the prosegment is known to occur in cis for these precursor molecules.

The transcriptional corepressor MITR is a signal-responsive inhibitor of myogenesis

Activation of muscle-specific genes by members of the myocyte enhancer factor 2 (MEF2) and MyoD families of transcription factors is coupled to histone acetylation and is inhibited by class II histone deacetylases (HDACs) 4 and 5, which interact with MEF2. The ability of HDAC4 and -5 to inhibit MEF2 is blocked by phosphorylation of these HDACs at two conserved serine residues, which creates docking sites for the intracellular chaperone protein 14-3-3. When bound to 14-3-3, HDACs are released from MEF2 and transported to the cytoplasm, thereby allowing MEF2 to stimulate muscle-specific gene expression. MEF2-interacting transcription repressor (MITR) shares homology with the amino-terminal regions of HDAC4 and -5, but lacks an HDAC catalytic domain. Despite the absence of intrinsic HDAC activity, MITR acts as a potent inhibitor of MEF2-dependent transcription. Paradoxically, however, MITR has minimal inhibitory effects on the skeletal muscle differentiation program. We show that a substitution mutant of MITR containing alanine in place of two serine residues, Ser-218 and Ser-448, acts as a potent repressor of myogenesis. Our findings indicate that promyogenic signals antagonize the inhibitory action of MITR by targeting these serines for phosphorylation. Phosphorylation of Ser-218 and Ser-448 stimulates binding of 14-3-3 to MITR, disrupts MEF2:MITR interactions, and alters the nuclear distribution of MITR. These results reveal a role for MITR as a signal-dependent regulator of muscle differentiation.