Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells.

We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.

Human 92 kDa type IV collagenase: functional analysis of fibronectin and carboxyl-end domains.

Two closely related secreted metalloproteases 72 and 92 kDa type IV collagenases (72- and 92T4Cl) consist of several structural domains, the functions of which are poorly understood. Both metalloproteases can bind to gelatin as well as form complexes with specific inhibitors in the proenzyme form. The biologic role of the proenzyme-inhibitor complex formation remained unclear. Here we summarize results demonstrating that the fibronectin-like domain of 92T4Cl mediates gelatin binding of the proenzyme, while the hemopexin like carboxy-terminal domain is essential for the complex formation of the proenzyme with TIMP. The formation of a 92T4Cl proenzyme complex with TIMP prevents dimerization, formation of the novel complex with ClI proenzyme, and activation of the 92T4Cl by stromelysin. Conversely, formation of the covalent 92T4Cl homodimer excludes the formation of a proenzyme-TIMP complex, thus allowing this form of enzyme to enter into the proteolytic cascade of activation. Both components of the 92T4Cl-ClI complex can be activated in a fashion similar to that of free enzymes, yielding a complex active against both gelatin and fibrillar collagen.

Alanine scanning mutagenesis and functional analysis of the fibronectin-like collagen-binding domain from human 92-kDa type IV collagenase.

The human 72-kDa (CLG4A) and 92-kDa (CLG4B) type IV collagenases contain a domain consisting of three contiguous copies of the fibronectin (FN)-derived type II homology unit (T2HU), T2HU-1, T2HU-2, and T2HU-3. To investigate the functional role of this domain, we have constructed plasmids expressing beta-galactosidase fusion proteins with one or more of the CLG4B-derived T2HU. The gelatin binding assays demonstrate that a single copy of T2HU-2 renders beta-galactosidase capable of binding gelatin. The three repeats, however, differ dramatically in their capacity to bind gelatin, with T2HU-1 and T2HU-3 having significantly less binding activity than T2HU-2. Using alanine scanning mutagenesis we have defined the amino acid residues (Arg307, Asp309, Asn319, Tyr320, Asp323) that are critical for gelatin binding of T2HU-2. The low gelatin binding of T2HU-1 compared to T2HU-2 was traced to the non-conserved residues Ala228-Ala and Leu253-Pro. The results suggest that the gelatin binding of the type IV collagenase proenzyme is mediated by the FN-like domain, although the presence of another gelatin-binding site cannot be excluded. The FN domain-mediated binding, however, is not a rate-limiting step in the hydrolysis of gelatin by the enzyme.

[Difference in the protein composition of human leukocytes normally and in aneuploidy]. / O razlichiiakh belkovogo sostava leikotsitov cheloveka v norme i pri aneuploidii.

Using polyacrylamide gel electrophoresis an additional protein band was found in the leukocyte preparation obtained from patients with Down syndrome as compared with the cells of healthy donors. Impairments in gene expression, due to alteration in the ratio between content of histone genes and of nuclear DNA, were apparently responsible for qualitative alterations in the cell protein composition under conditions of aneuploidy as well as for the quantitative alterations, deviating from the principle of "gene dosage".

[Dissociation and decompactization of chromatin by heparin in a medium of "physiologic" ionic strength]. / Dissotsiatsiia i dekompaktizatsiia khromatina geparinom v srede "fiziologicheskoi"ionnoii sily.

It has been shown for the first time that heparin, one of the natural polyanions, is capable of dissociating the histones H1, H2A and H2B from chromatin in a medium of "physiological" ionic strength: 0.15 M NaCl + 0.7 mM Na-phosphate buffer, pH 7.0. Under the same conditions heparin produces chromatin fractionation, leading to the appearance of slow-sedimenting DNA, augments viscosity and reduces turbidity of chromatin suspension. This evidences the decompactization of deoxyribonucleoprotein fibrils.