The catalytic role of the active site aspartic acid in serine proteases.

The role of the aspartic acid residue in the serine protease catalytic triad Asp, His, and Ser has been tested by replacing Asp102 of trypsin with Asn by site-directed mutagenesis. The naturally occurring and mutant enzymes were produced in a heterologous expression system, purified to homogeneity, and characterized. At neutral pH the mutant enzyme activity with an ester substrate and with the Ser195-specific reagent diisopropylfluorophosphate is approximately 10(4) times less than that of the unmodified enzyme. In contrast to the dramatic loss in reactivity of Ser195, the mutant trypsin reacts with the His57-specific reagent, tosyl-L-lysine chloromethylketone, only five times less efficiently than the unmodified enzyme. Thus, the ability of His57 to react with this affinity label is not severely compromised. The catalytic activity of the mutant enzyme increases with increasing pH so that at pH 10.2 the kcat is 6 percent that of trypsin. Kinetic analysis of this novel activity suggests this is due in part to participation of either a titratable base or of hydroxide ion in the catalytic mechanism. By demonstrating the importance of the aspartate residue in catalysis, especially at physiological pH, these experiments provide a rationalization for the evolutionary conservation of the catalytic triad.

Redesigning trypsin: alteration of substrate specificity.

A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.

The HOX homeodomain proteins block CBP histone acetyltransferase activity.

Despite the identification of PBC proteins as cofactors that provide DNA affinity and binding specificity for the HOX homeodomain proteins, HOX proteins do not demonstrate robust activity in transient-transcription assays and few authentic downstream targets have been identified for these putative transcription factors. During a search for additional cofactors, we established that each of the 14 HOX proteins tested, from 11 separate paralog groups, binds to CBP or p300. All six isolated homeodomain fragments tested bind to CBP, suggesting that the homeodomain is a common site of interaction. Surprisingly, CBP-p300 does not form DNA binding complexes with the HOX proteins but instead prevents their binding to DNA. The HOX proteins are not substrates for CBP histone acetyltransferase (HAT) but instead inhibit the activity of CBP in both in vitro and in vivo systems. These mutually inhibitory interactions are reflected by the inability of CBP to potentiate the low levels of gene activation induced by HOX proteins in a range of reporter assays. We propose two models for HOX protein function: (i) HOX proteins may function without CBP HAT to regulate transcription as cooperative DNA binding molecules with PBX, MEIS, or other cofactors, and (ii) the HOX proteins may inhibit CBP HAT activity and thus function as repressors of gene transcription.

Pbx modulation of Hox homeodomain amino-terminal arms establishes different DNA-binding specificities across the Hox locus.

Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, including class I Hox proteins. To assess how Pbx proteins influence Hox DNA-binding specificity, we used a binding-site selection approach to determine high-affinity target sites recognized by various Pbx-Hox homeoprotein complexes. Pbx-Hox heterodimers preferred to bind a bipartite sequence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) and the Hox component contacted the more variable 3' half (TNATNN). Binding sites matching the consensus were also obtained for Pbx1 complexed with HoxA10, which lacks a hexapeptide but requires a conserved tryptophan-containing motif for cooperativity with Pbx. Interactions with Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish single nucleotide alterations in the Hox half site both in vitro and in cellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their preferences for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes in the Hox cluster. These observations demonstrate previously undetected DNA-binding specificity for the amino-terminal arm of the Hox homeodomain and suggest that different binding activities of Pbx-Hox complexes are at least part of the position-specific activities of the Hox genes.

HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells.

Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets.

Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia.

Multiple members of the A, B, and C clusters of Hox genes are expressed in hematopoietic cells. Several of these Hox genes have been found to display distinctive expression patterns, with genes located at the 3' side of the clusters being expressed at their highest levels in the most primitive subpopulation of human CD34+ bone marrow cells and genes located at the 5' end having a broader range of expression, with downregulation at later stages of hematopoietic differentiation. To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4. In contrast to HOXB4, which causes selective expansion of primitive hematopoietic cells without altering their differentiation, overexpression of HOXA10 profoundly perturbed myeloid and B-lymphoid differentiation. The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contained in high frequency a unique progenitor cell with megakaryocytic colony-forming ability and was virtually devoid of unilineage macrophage and pre-B-lymphoid progenitor cells derived from the transduced cells. Moreover, and again in contrast to HOXB4, a significant proportion of HOXA10 mice developed a transplantable acute myeloid leukemia with a latency of 19 to 50 weeks. These results thus add to recognition of Hox genes as important regulators of hematopoiesis and provide important new evidence of Hox gene-specific functions that may correlate with their normal expression pattern.

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.

Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with PBX1.

The products of PBX homeobox genes, which were initially discovered in reciprocal translocations occurring in human leukemias, have been shown to cooperate in the in vitro DNA binding with HOX proteins. Despite the growing body of data implicating Hox genes in the development of various cancers, little is known about the role of HOX-PBX interactions in the regulation of proliferation and induction of transformation of mammalian cells. We build on the existing model of Hox-induced transformation of Rat-1 cells to show that both cellular transformation and proliferation induced by Hoxb4 and Hoxb3 are greatly modulated by the levels of available PBX1 present in these cells. Furthermore, we show that the transforming capacity of these two HOX proteins depends on their conserved tetrapeptide and homeodomain regions which mediate binding to PBX and DNA, respectively. Taken together, results of this study demonstrate that cooperation between HOX and PBX proteins modulates cellular proliferation and strongly suggest that cooperative DNA binding by these two groups of proteins represent the basis for Hox-induced cellular transformation.

Human pancreatic proelastase 2. Sequence of the activation peptide.

The N-terminal sixteen residues of the amino acid sequence of reduced and alkylated human pancreatic proelastase 2 have been established, and N-terminal amino acid residue has been shown to be carboxymethylcysteine. A peptide containing an amino acid sequence corresponding to the first twelve residues of proelastase 2 was isolated following activation of proelastase 2 with trypsin, performic acid oxidation, and gel filtration. This peptide was not released prior to performic acid oxidation, suggesting that it remains attached to the major peptide chain via a disulfide bond containing the N-terminal half-cysteine in a manner similar to that found for chymotrypsinogens. However, the amino acid sequence of the activation peptide is not strongly homologous to either porcine chymotrypsinogen A or porcine proelastase 1.

Human carboxypeptidase B. II. Purification of the enzyme from pancreatic tissue and comparison with the enzymes present in pancreatic secretion.

Carboxypeptidase B (peptidyl-L-lysine (-L-arginine) hydrolase, EC 3.4.12.3) has been isolated and purified to apparent homogeneity from activated extracts of human pancreas tissue. The purified enzyme has been shown to be a single polypeptide of 34 000 daltons. In this respect the enzyme from pancreatic tissue, designated native human carboxypeptidase B, differs from the two forms present in human pancreatic juice (fractions I and II), both of which are composed of two polypeptides of approximately 24 000 and 9000 daltons. In addition, the three forms of human carboxypeptidase B differ in electrophoretic mobility in polyacrylamide gel electrophoresis and in chromatographic behavior on DEAE-cellulose. Two immunological methods, micro-complement fixation and radioimmunoassay, have shown a high degree of structural similarity between the three forms of human carboxypeptidase B. Micro-complement fixation experiments indicate that the amino acid sequences of the three enzymes differ by less than one percent. In vitro digestion studies have indicated that trypsin alone is sufficient to convert native carboxypeptidase B to carboxypeptidase B II. However, no combination of trypsin, chymotrypsin, and/or elastase was capable of converting native carboxypeptidase B to carboxypeptidase B I in vitro.

Human pancreatic carboxypeptidase B. I. Isolation, purification, and characterization of fraction II.

Human carboxypeptidase B fraction II has been purified from pancreatic juice by DEAE-'Sephadex' chromatography, isoelectric focusing, and 'Sephadex' G-100 gel filtration. The enzyme has been characterized by analytical polyacrylamide disc-gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid analysis Km determination, molecular weight determination on 'Sephadex' G-100, zinc analysis, and inhibition by metal chelating agents. Human carboxypeptidase B fraction II appeared homogeneous in analytical polyacrylamide disc-gel electrophoresis, but showed two components of 23,500 and 9,200 daltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Zinc analysis revealed 0.96 gram atoms of zinc per mole of enzyme, and a Km of 65 +/- 3 muM was determined for hydrolysis of hippuryl-L-arginine.

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay.

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors alpha1-antitrypsin and alpha2-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.

Inhibition of human pancreatic elastase 2 by peptide chloromethyl ketones.

The inactivation of human pancreatic elastase 2 (EC 3.4.21.11) by a series of peptide chloromethyl ketones has been investigated. Among a series of compounds with the structure X-Ala-Ala-Pro-Y-CH2Cl (where X=acetyl-, succinyl-, methylsuccinyl-, or H-), the kinetic parametrs for inhibition of elastas 2 depend markedly on the amino acid (Y) in the P1 position. Succinyl-Ala-Ala-Pro-Leu-CH2Cl was found to be an extremely effective inhibitor of human elastase 2, qith a first-order rate constant for covalent bond formation (k3) of 0.033s-1 and a dissociation constant, Ki, for the enzyme inhibitor complex of 7.4 . 10(-7) M. The second-order rate constant k3/Ki for inhibition of elastase 2 by the analogous compound containing a free amino group in place of the succinyl moiety is 150 times lower than that found for the succinyl or acetyl derivative, suggesting that the presence of a positive charge at this position reduces the proper binding of the inhibitor to the enzyme.

Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias.

There is increasing evidence that HOX homeobox genes play a role in leukemogenesis. Recent studies have demonstrated that enforced co-expression of HOXA9 and MEIS1 in murine marrow leads to rapid development of myeloid leukemia, and that these proteins exhibit cooperative DNA binding. However, it is unclear whether co-activation of HOXA9 and MEIS genes is a common occurrence in human leukemias. We surveyed expression of HOXA9 and MEIS1 in 24 leukemic cell lines and 80 patient samples, using RNase protection analyses and immunohistochemistry. We demonstrate that the expression of HOXA9 and MEIS1 in leukemia cells is uniquely myeloid, and that these genes are commonly co-expressed in myeloid cell lines and in samples of acute myelogenous leukemia (AML) of all subtypes except in promyelocytic leukemia. While HOXA9 is expressed in most cases of chronic myelogenous leukemia, MEIS1 is weakly expressed or not at all. Immunohistochemical staining of selected AML samples showed moderate to high levels of HOXA9 protein, primarily cytoplasmic, in leukemic myeloblasts, with weaker and primarily nuclear staining for MEIS1. These data support the concept that co-activation of HOXA9 and MEIS1 is a common event in AML, and may represent a common pathway of many different oncogenic mutations.

Stage- and lineage-specific expression of the HOXA10 homeobox gene in normal and leukemic hematopoietic cells.

There is growing evidence that the HOX homeobox-containing transcription factors are differentially expressed during hematopoiesis. We have previously demonstrated that the HOXA10 gene is expressed in unfractionated normal marrow and in immortalized leukemic cell lines with myelomonocytic features, but not in cell lines with lymphoid or erythroid features. To gain insights into the patterns of activation of this gene during hematopoietic differentiation, we have examined HOXA10 expression in CD34+ and CD34- subfractions of normal marrow and normal peripheral blood, as well as samples from patients with a variety of acute and chronic leukemias. HOXA10 is strongly expressed in CD34+ normal marrow cells, markedly downregulated in CD34- marrow cells, and inactive in mature neutrophils, monocytes, and lymphocytes. HOXA10 is expressed in all types of acute myelogenous leukemia (AML) with the notable exception of acute promyelocytic leukemia (AML-M3). HOXA10 message is observed in chronic myelogenous leukemia (CML) but appears to be reduced in accelerated phase and blast crisis, particularly lymphoid blast crisis. With rare exception, HOXA10 expression is not observed in samples of acute or chronic lymphoid leukemias. Normal marrow and patient samples appear to contain a single transcript which encodes a full-length homeobox-containing protein, while immortalized cell lines contain an additional alternatively spliced transcript. These studies indicate that HOXA10 expression is restricted to early stages of myeloid differentiation.

Expression of class I homeobox genes in fetal and adult murine skin.

We examined the expression patterns of several class I homeobox genes in mouse fetal and adult skin. All the genes of the Hox-B locus, except Hoxb-1, are expressed in skin from murine fetuses of 17 and 18 d gestation, at which time the epidermis is undergoing stratification and differentiation. The amount of individual Hox gene message varies considerably, but expression of all genes is detectable by RNase protection except Hoxb-1, which could not be detected even by the reverse transcription-polymerase chain reaction (RT-PCR) assay. Homeobox gene expression in skin is not confined to the Hox-B locus; the paralogous genes Hoxa-4, -b-4, -c-4, and -d-4 are all expressed. The amount of Hoxb-4, -b-2, and -c-4 message in skin is relatively constant from the earliest gestational day examined (day 16) through birth at day 19. Expression of several homeobox genes is also seen in adult skin.

Modulation of the human homeobox genes PRX-2 and HOXB13 in scarless fetal wounds.

Scarless healing of cutaneous wounds occurs in humans during the first two trimesters of development, but by birth all wounds are repaired with scar formation. To search for transcriptional regulatory genes that might mediate fetal tissue regeneration, we surveyed homeobox gene expression in proliferating fetal fibroblasts and in wounded and unwounded skin. Two novel human homeobox genes, PRX-2 and HOXB13, were identified that were differentially expressed during fetal versus adult wound healing. Both genes were predominantly expressed in proliferating fetal fibroblasts and developing dermis, and PRX-2 was downregulated in adult skin. In a model of scarless fetal skin regeneration, PRX-2 expression was strongly increased compared with unwounded skin and the signal was localized to the wounded dermis, the site of scarless repair. Conversely, in adult skin weak epidermal PRX-2 expression was observed, mRNA levels were not increased by wounding, and no dermal expression was detected. HOXB13 expression was decreased in wounded fetal tissue relative to unwounded fetal controls or wounded adult skin. Thus both HOXB13 and PRX-2 are expressed in patterns consistent with roles in fetal skin development and cutaneous regeneration.

Epithelial sodium channels are upregulated during epidermal differentiation.

Terminal differentiation of epidermal keratinocytes is linked to transmembrane ion flux. Previously, we have shown that amiloride, an inhibitor of epithelial sodium channels, blocks synthesis of differentiation-specific proteins in normal human keratinocytes. Here, we have identified the specific subunits of amiloride-sensitive human epithelial sodium channels in relation to differentiation of cultured human keratinocytes, as well as to epidermal development. As assessed by northern hybridization, RNase protection assay, and reverse transcription-polymerase chain reaction, transcripts encoding functional alpha and regulatory beta subunits of human epithelial sodium channels were expressed both in cultured keratinocytes and in epidermis at levels comparable with the kidney. The mRNA expression of both human epithelial sodium channel-alpha and -beta increased during calcium-induced keratinocyte differentiation. Whereas the beta subunit of human epithelial sodium channel was induced by elevated concentrations of calcium, the alpha subunit increased with duration of culture. The regulatory gamma subunit was less abundant but also expressed in epidermis. Both human epithelial sodium channel-alpha and -beta were localized throughout the nucleated layers of human adult epidermis, but these channels were not detected in early stages of fetal epidermal development. This co-ordinated expression of subunits suggests that epithelial sodium channels may play an important part in both epidermal differentiation and skin development, presumably by modulating ion transport required for epidermal terminal differentiation.

HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.

The spatial and temporal deployment of HOX homeobox genes along the spinal axis and in limb buds during fetal development is a key program in embryonic pattern formation. Although we have previously reported that several of the HOX homeobox genes are expressed during murine skin development, there is no information about developmental expression of HOX genes in human skin. We have now used reverse transcriptase polymerase chain reaction, in conjunction with a set of degenerate oligonucleotide primers, to identify a subset of HOX genes that are expressed during human fetal skin development. In situ hybridization analyses demonstrated that there were temporal and spatial shifts in expression of these genes. Strong HOXA4 expression was detected in the basal cell layers of 10 wk fetal epidermis and throughout the epidermis and dermis of 17 wk skin, whereas weak signal was present in the granular layer of newborn and adult skin. The expression patterns of HOXA5 and HOXA7 were similar, but their expression was weaker. In situ hybridization analysis also revealed strong HOXC4 and weaker HOXB7 expression throughout fetal development, whereas HOXB4 was expressed at barely detectable levels. Differential HOX gene expression was also observed in developing hair follicles, and sebaceous and sweat glands. None of the HOX genes examined were detected in the adult dermis.

Alternatively spliced forms of the cGMP-gated channel in human keratinocytes.

Alternatively spliced forms of the alpha subunit of the cGMP-gated channel have been cloned from human keratinocytes. One form encodes a complete channel which is almost identical to the rod photoreceptor. A second spliced variant would encode a protein missing a portion of the intracellular hydrophilic domain and the putative first transmembrane domain. Both complete and spliced variants of the channel also were found in epidermis. The expression of the complete form of the channel was induced by levels of extracellular calcium which promote keratinocyte differentiation. The cGMP-gated channel may play an important role in calcium induced keratinocyte differentiation by mediating Ca2+ entry.