Differential roles of TIMP-4 and TIMP-2 in pro-MMP-2 activation by MT1-MMP

The tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of MMP enzymatic activity. However, TIMP-2 can promote the activation of pro-MMP-2 by MT1-MMP. This process is mediated by the formation of a complex between MT1-MMP, TIMP-2, and pro-MMP-2. Binding of TIMP-2 to active MT1-MMP also inhibits the autocatalytic turnover of MT1-MMP on the cell surface. Thus, under certain conditions, TIMP-2 is a positive regulator of MMP activity. TIMP-4, a close homologue of TIMP-2 also binds to pro-MMP-2 and can potentially participate in pro-MMP-2 activation. We coexpressed MT1-MMP with TIMP-4 and investigated its ability to support pro-MMP-2 activation. TIMP-4, unlike TIMP-2, does not promote pro-MMP-2 activation by MT1-MMP. However, TIMP-4 binds to MT1-MMP inhibiting its autocatalytic processing. When coexpressed with TIMP-2, TIMP-4 competitively reduced pro-MMP-2 activation by MT1-MMP. A balance between TIMP-2 and TIMP-4 may be a critical factor in determining the degradative potential of cells in normal and pathological conditions.     

Glycosylation broadens the substrate profile of membrane type 1 matrix metalloproteinase

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme that has been implicated in normal development and in pathological processes such as cancer metastasis. The activity of MT1-MMP is regulated extensively at the post-translational level, and the current data support the hypothesis that MT1-MMP activity is modulated by glycosylation. Enzymatic deglycosylation, site-directed mutagenesis, and lectin precipitation assays were used to demonstrate that MT1-MMP contains O-linked complex carbohydrates on the Thr(291), Thr(299), Thr(300), and/or Ser(301) residues in the proline-rich linker region. MT1-MMP glycoforms were detected in human cancer cell lines, suggesting that MT1-MMP activity may be regulated by differential glycosylation in vivo. Although the autolytic processing and interstitial collagenase activity of MT1-MMP were not impaired in glycosylation-deficient mutants, cell surface MT1-MMP-catalyzed activation of pro-matrix metalloproteinase-2 (proMMP-2) required proper glycosylation of MT1-MMP. The inability of carbohydrate-free MT1-MMP to activate proMMP-2 was not a result of defective MT1-MMP zymogen activation, aberrant protein stability, or inability of the mature enzyme to oligomerize. Rather, our data support a mechanism whereby glycosylation affects the recruitment of tissue inhibitor of metalloproteinases-2 (TIMP-2) to the cell surface, resulting in defective formation of the MT1-MMP/TIMP-2/proMMP-2 trimeric activation complex. These data provide evidence for an additional mechanism for post-translational control of MT1-MMP activity and suggest that glycosylation of MT1-MMP may regulate its substrate targeting.     

Deletion of the Tail Domain of the Kinesin-5 Cin8 Affects Its Directionality

The bipolar kinesin-5 motors are one of the major players that govern mitotic spindle dynamics. Their bipolar structure enables them to cross-link and slide apart antiparallel microtubules (MTs) emanating from the opposing spindle poles. The budding yeast kinesin-5 Cin8 was shown to switch from fast minus-end- to slow plus-end-directed motility upon binding between antiparallel MTs. This unexpected finding revealed a new dimension of cellular control of transport, the mechanism of which is unknown. Here we have examined the role of the C-terminal tail domain of Cin8 in regulating directionality. We first constructed a stable dimeric Cin8/kinesin-1 chimera (Cin8Kin), consisting of head and neck linker of Cin8 fused to the stalk of kinesin-1. As a single dimeric motor, Cin8Kin switched frequently between plus and minus directionality along single MTs, demonstrating that the Cin8 head domains are inherently bidirectional, but control over directionality was lost. We next examined the activity of a tetrameric Cin8 lacking only the tail domains (Cin8Δtail). In contrast to wild-type Cin8, the motility of single molecules of Cin8Δtail in high ionic strength was slow and bidirectional, with almost no directionality switches. Cin8Δtail showed only a weak ability to cross-link MTs in vitro. In vivo, Cin8Δtail exhibited bias toward the plus-end of the MTs and was unable to support viability of cells as the sole kinesin-5 motor. We conclude that the tail of Cin8 is not necessary for bidirectional processive motion, but is controlling the switch between plus- and minus-end-directed motility.     

Experimental (preclinical) study of genetically engineered human interferon based on recombinant clones of E. coli 74 and Pseudomonas Sp VG-84

The results of the experimental (preclinical) study of interferon alpha2 from Pseudomonas sp VG-84 obtained from the All-Union Research Institute of Genetics and Selection of Industrial Microorganisms in comparison with the preparations of interferon alpha2 from E. coli 74 and natural human interferon (obtained from the Gamaleya Institute of Epidemiology and Microbiology) are presented. Interferon alpha2 from Pseudomonas, a highly purified preparation, has been shown to possess pronounced biological activity (antiviral and antiproliferative) in the culture of human cells. The medicinal form of the preparation has proved to be completely safe and areactive for animals, which makes it possible to recommend the preparation for testing on volunteers.     

Cellular immune reactions to the multiple administration of a chromatographic inactivated influenza vaccine in an experiment

The administration of inactivated chromatographic influenza vaccine to mice in three injections induced the formation of the pronounced clone of antigen-reactive lymphocytes, detected in the leukocyte blast transformation test. Slight fluctuations in the phytohemagglutinin level and lipopolysaccharide response in mice subjected to multiple immunization with the inactivated vaccine indicated that this preparation produced no damaging effect on the T- and B-lymphocyte populations.     

Increased expression of perforin and granzyme B genes in patients with metastatic melanoma treated with recombinant interleukin-2

The frequency of peripheral blood cells expressing the perforin gene or the granzyme B gene was evaluated by in situ hybridization in nine patients suffering from metastatic melanoma and treated with recombinant interleukin-2 (rIL-2). A spontaneous expression of both genes was detected in five to seven patients, rIL-2 administration increased the frequency of positive cells in all patients (P<0.03 for each gene), the highest frequency being reached in the patients who already expressed these genes prior to rIL-2 treatment (P<0.02). Expressions of the granzyme B gene and of the perforin gene were strongly correlated before IL-2 treatment and they were similarly affected by rIL-2 administration. In contrast, their modification under treatment did not correlate with that of CD56⁺ cell counts, of natural killer activity and of sCD8 release. This indicates that perforin and granzyme B gene expressions are markers of cytotoxic cell activation independent of those previously described, and that they should be further evaluated in patients with malignancies to delineate their potential value in predicting clinical outcome.     

Dissociation kinetics of an enzyme-inhibitor system using single-molecule force measurements

We report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme-inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s(-1) for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.