Jeffrey A. Engler

Matrix Metalloproteinases: A Review

Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn(++)-endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. The present review discusses in detail the primary structures and the overlapping yet distinct substrate specificities of MMPs as well as the mode of activation of the unique MMP precursors. The regulation of MMP activity at the transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes) is also discussed. A final segment of the review details the current knowledge of the involvement of MMP in specific developmental or pathological conditions, including human periodontal diseases.

Receptor-mediated uptake of peptides that bind the human transferrin receptor

A biopanning process designed to find peptide epitopes specific for cell surface receptors has been used in this study to select seven- and 12-amino-acid peptides capable of binding to and internalizing with the human transferrin receptor (hTfR). Through sequential rounds of negative and positive selection, two peptide sequences were identified that specifically bind to the hTfR. Phage containing the sequences HAIYPRH or THRPPMWSPVWP were inhibited from binding the hTfR in a dose-dependent fashion when peptides of the same sequence were present in a competition assay. Interestingly, transferrin did not compete with either of these sequences for receptor binding, suggesting that these peptides bind a site on the hTfR distinct from the transferrin binding site. When either of these sequences was expressed as a fusion to green fluorescent protein (GFP), the recombinant GFP molecule was internalized in cells expressing the hTfR. These studies suggest that the two peptides can be used to target other proteins into the endosomal pathway. Further, they provide a strategy for identifying peptides that bind to other cell surface receptors that can be used for both diagnostic and therapeutic purposes.

Structural models of human apolipoprotein A-I: a critical analysis and review

Human apolipoprotein (apo) A-I has been the subject of intense investigation because of its well-documented anti-atherogenic properties. About 70% of the protein found in high density lipoprotein complexes is apo A-I, a molecule that contains a series of highly homologous amphipathic alpha-helices. A number of significant experimental observations have allowed increasing sophisticated structural models for both the lipid-bound and the lipid-free forms of the apo A-I molecule to be tested critically. It seems clear, for example, that interactions between amphipathic domains in apo A-I may be crucial to understanding the dynamic nature of the molecule and the pathways by which the lipid-free molecule binds to lipid, both in a discoidal and a spherical particle. The state of the art of these structural studies is discussed and placed in context with current models and concepts of the physiological role of apo A-I and high-density lipoprotein in atherosclerosis and lipid metabolism.

Nuclear Localization of KLF4 Is Associated with an Aggressive Phenotype in Early-Stage Breast Cancer

Purpose: The Krüppel-like transcription factor KLF4/GKLF induces both malignant transformation and a slow-growth phenotype in vitro. Although KLF4 expression is increased in most cases of breast cancer, it was unknown whether these cases represent a distinct subtype with a different clinical outcome. Experimental Design: We examined expression of KLF4 by immunostaining 146 cases of human primary infiltrating ductal carcinoma of the breast. Staining patterns were correlated with clinical outcome and with established prognostic factors. Results: Subcellular localization exhibited case-to-case variation. Tumors with high nuclear staining and low cytoplasmic staining were termed type 1. For patients with early-stage disease (i.e., stage I or IIA), type 1 staining was associated with eventual death because of breast cancer (hazard ratio, 2.8; 95% confidence interval, 1.23–6.58; P = 0.011). The association was stronger in patients with early-stage cancer and small primary tumors (i.e., ≤2.0 cm in diameter; hazard ratio, 4.3; 95% confidence interval, 1.75–10.62; P < 0.001). For patients with early-stage disease, multivariate analysis indicated that type 1 staining was independently associated with outcome (adjusted hazard ratio 2.6; 95% confidence interval, 1.10–6.05; P = 0.029). Type 1 staining was also associated with high histological grade (P = 0.032), increased expression of Ki67 (P = 0.016), and reduced expression of BCL2 (P = 0.032). In vitro, KLF4 was localized within the nucleus of transformed RK3E epithelial cells, consistent with a nuclear function of this transcription factor during induction of malignant transformation. Conclusions: The results suggest that localization of KLF4 in the nucleus of breast cancer cells is a prognostic factor and identify KLF4 as a marker of an aggressive phenotype in early-stage infiltrating ductal carcinoma.

The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30.

A group B streptococcal (GBS) bacteriophage lysin gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other beta-haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including Streptococcus mutans. The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.

The amino terminus of the adenovirus fiber protein encodes the nuclear localization signal.

Using a recombinant vaccinia virus vector, the fiber protein from adenovirus serotype 2 has been expressed in human cells; the protein expressed was correctly assembled into trimers, glycosylated, and transported to the nucleus. Deletion of amino acids 2-5 (KRAR) resulted in accumulation of fiber in the cytoplasm; fusion of the sequence TKRVRL, found at the beginning of Ad7 fiber, to the N-terminus of this mutant restored correct targeting. Changing the charge of amino acids 91 and 92 within another potential targeting sequence (LKKTK to LEETK) had little effect on nuclear targeting. When fused to the N-terminus of beta-galactosidase and expressed in recombinant vaccinia virus, neither MKRARP nor MTKRVRL (from Ad2 and Ad7 fibers, respectively), were sufficient for efficient transport of the hybrid protein to the nucleus; on the other hand, fusions of either MKRARPSEDTF (from Ad2 fiber) or of MKRPRP (a known targeting sequence from the C-terminus of Ad2 E1A proteins) to beta-galactosidase were localized to the nucleus. These results suggest that sequences at the N-terminus of Ad2 and Ad7 fiber are required for correct nuclear targeting.

Induction of collagenase-3 (MMP-13) in rheumatoid arthritis synovial fibroblasts.

There is a growing body of evidence that implicates matrix metalloproteinases (MMPs) as major players in numerous diseased conditions. The articular cartilage degradation that is characteristic of rheumatoid arthritis (RA) is believed to be mediated by the collagenase subfamily of matrix metalloproteinases. The preference of collagenase-3 (CL-3) for collagen type II makes it a likely candidate in the turnover of articular cartilage and a potential target for drug development. In this study, RA synovial membrane tissue was shown to express CL-3 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunohistochemistry. Fibroblasts isolated and cultured from RA synovial membrane tissue were induced to express CL-3 mRNA. CL-3 mRNA was detected after PMA treatment in 16 of the 18 RA synovial membrane fibroblast cell lines established for this study. These fibroblasts also expressed mRNA for collagenase-1 (CL-1, MMP-1), membrane type-1 matrix metalloproteinase, gelatinase A, gelatinase B, stromelysin-1, stromelysin-2, TIMP-1, and TIMP-2. They were further shown to express CL-1 mRNA constitutively and CL-3 mRNA only after stimulation with PMA, IL-1, TGF-beta1, TNF-alpha, or IL-6 with IL-6sR. These fibroblasts also expressed after induction both CL-1 and CL-3 at the protein level as determined by Western blot analyses and immunofluorescence.

Group b streptococcal phage lysin

Two stages of phage multiplications, the adsorption-penetration process and the lysis of the host, have recently been largely elucidated. For some time it has been postulated that an enzyme attached to, or contained in, the phage tail is involved in the adsorption process with at least some phage-host systems. Similarly, the earliest discovered effect of a lytic phage, namely the liberation of the mature phage particles (lysis of the host), is due to a lytic enzyme acting on the cell wall. Bronfenbrenner & Muckenfuss (1927) recognized that phage lysates contain a substance other than phage. This ‘ ferment-like ’ substance differed from phage because it only affected dead staphylococci. It was adsorbed to clay filters, inactivated on standing at room temperature, it was more heat labile than phage, and did not diffuse through collodion membranes. This description adequately fits the staphylococcal virolysin (or phage lysin) described by Ralston, Beer, Lieberman & Krueger (1955) which lysed only dead cells, although viable cells were lysed in the presence of the homologous phage. Brown (1956) showed that phage-free lysates of coliphage T, could dissolve cells of Eschterichia coli killed with chloroform, and Koch & Jordan (1957) demonstrated a free enzyme in T, phage lysates. Koch & Dreyer (1958) came to the conclusion that the phage lysin was a lysozyme. This was confirmed by Weidel & Katz (1961) and K,atz & Weidel (1961) who purified both the bound and free coli T, phage enzymes; and Murphy (1960), working on similar lines, found that the bound and free ‘ megaterium ’ enzymes differed only in their pH optima. Halo1 formation was observed by Naylor & Czulak (1956) with a lactic acid streptococcal phage, and by Murphy (1957; 1960) with Bacillus megaterium phage. They found that halos were due to an enzyme affecting the dead cells surrounding the phages without giving complete lysis. Murphy demonstrated that the enzyme was resistant to trypsin, pepsin, DNA-ase and RNA-ase but was inactivated by chymotrypsin. It is possible that the inhibition of a rhizobium phage by chymotrypsin reported by Kleczkowsky & Kleczkowsky (1954) may be due to the inhibition of a similar phage enzyme. Halo formation due to cell-wall lysis differs from the halos observed around plaques of mucoid strains of Escherichia coli by Sertic (1929), and since reported to occur with many other organisms. In these cases the halo is due to an enzyme hydrolysing capsular polysaccharides (Adams & Park, 1956). Other aspects of the action of phage lysin are the ‘nascent’ phage phenomenon and ‘lysis from without’. Nascent phage was first observed by Evans (1934) and shown by Maxted (1957) to be due to an enzyme liberated from the host at the same time as the phage. When some phages, e.g. coliphage T,, are adsorbed to their host

Nucleotide sequence of the genes encoded in early region 2b of human adenovirus type 12

The nucleotide (nt) sequence of a cloned DNA segment containing the early 2b region of the class A adenovirus Ad12 has been determined. When compared to the corresponding region of Ad2 or Ad7, there is a high degree of nt and predicted amino acid (aa) sequence homology within the r-strand regions that encode the preterminal protein and the viral DNA polymerase. A gene coding region comparable to the Mr 13,600 gene product found in Ad2 can be identified; this hypothetical gene product shares 30% aa homology with its Ad2 counterpart and has a very similar hydropathy profile.

Mutagenesis of conserved region I in the DNA polymerase from human adenovirus serotype 2

The functional importance of the conserved region I (YGDTDSLF) found in several prokaryotic, eukaryotic, and viral DNA polymerases has been probed by site-directed mutagenesis of the adenovirus DNA polymerase (Ad Pol). Three different adenovirus-specific assays have been used to measure the in vitro activity of region I mutants of Ad Pol expressed in transiently transfected CMT-4 cells. In general, both conservative and nonconservative changes generally showed a greater than 5- to 10-fold reduction in activity in three different assays for activity. However, several replacements at the glycine residue showed activities closer to wild-type levels. For example, replacements of this glycine with cysteine (found in bacteriophage phi 29, another protein primed replication system), with serine, or with methionine had little effect on the activity observed in adenovirus-specific assays, such as initiation and elongation. These studies confirm the importance of this region of Ad Pol in specific initiation and elongation reactions on Ad DNA templates.