Biomedical subjects

Robert Janowski

Publications and source records attributed to Robert Janowski.

12 recordsLinked to original sources

PubMed2006

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of Rv2827c from Mycobacterium tuberculosis.

The hypothetical protein Rv2827c from Mycobacterium tuberculosis was cloned and heterologously expressed in Escherichia coli. It was purified using affinity and size-exclusion chromatographic techniques and then crystallized. Preliminary X-ray diffraction data analysis suggests the presence of two translationally related molecules in the asymmetric unit of the orthorhombic crystals.

Bacterial Proteins↗

PubMed2006

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: why is the L68Q variant amyloidogenic?

Human L68Q cystatin C is one of the known human amyloidogenic proteins. In its native state it is a monomer with alpha/beta structure. Experimental evidence suggests that L68Q variant associates into dimeric intermediates and that the dimers subsequently self-assemble to form amyloid deposits and insoluble fibrils. Details of the pathway of L68Q mutant amyloid formation are unclear; however, different experimental approaches with resolutions at molecular level have provided some clues. Probably, the stability and flexibility of monomeric L68Q variant play essential roles in the early steps of amyloid formation; thus, it is necessary to characterize early conformational changes of L68Q cystatin C monomers. In this paper, we demonstrate the possibility that the differences between the monomeric forms of wild-type (wt) cystatin C and its L68Q variant are responsible for higher tendency of the L68Q cystatin C amyloidogenesis. We started our studies with the simulations of wt and L68Q cystatin C monomers. Nanosecond time scale molecular dynamics simulations at 308K were performed using AMBER7.0 program. The results show that the structure of the L68Q monomer was changed, relative to the wt cystatin C structure. The results support earlier speculation that the L68Q point mutation would easily lead to dimer formation.

Amino Acid Substitution↗

PubMed2005

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets.

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N-truncated and full-length HCC (cubic form) showed dimer formation via three-dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain-swapping mechanism, but propagated in an open-ended fashion, could be the basis of HCC fibril formation. Here we report that full-length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The beta-strands of the beta-cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100 degrees in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular beta-sheets in an intermolecular context. At the other edge of the molecular beta-sheet, side-chain-side-chain hydrogen bonds propagate the beta-structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the beta-strands of the domain-swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X-ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so-called cross-beta structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite beta-sheets that are parallel to this axis.

Amyloid↗

PubMed2005

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapB (Rv2773c) from Mycobacterium tuberculosis.

Dihydrodipicolinate reductase from Mycobacterium tuberculosis (DapB, DHDPR, Rv2773c) has been cloned and heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in three different crystal forms. Preliminary diffraction data analysis suggests the presence of two tetramers in the asymmetric unit of one crystal form and half a tetramer in the other two crystal forms.

Anisotropy↗

PubMed2004

The high-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis.

The crystal structure of the enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (LeuB, Mtb-IPMDH, Rv2995c) without substrate or co-factor was determined at 1.65 A resolution, which is the highest resolution reported for an IPMDH to date. The crystals contain two functional dimers in the asymmetric unit in an arrangement close to a tetramer of D2 symmetry. Despite the absence of a substrate or inhibitor bound to the protein, the structure of the monomer resembles the previously observed closed form of the enzyme more closely than the open form. A comparison with the substrate complex of IPMDH from Thiobacillus ferrooxidans and the co-factor complex of the Thermus thermophilus enzyme revealed a close relationship of the active-site architecture between the various bacterial enzymes. The inhibitor O-isobutenyl oxalylhydroxamate was found to bind to the active site of IPMDH in a mode similar to the substrate isopropylmalate.

3-Isopropylmalate Dehydrogenase↗

PubMed2004

Domain swapping in N-truncated human cystatin C.

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.

Cystatin C↗

PubMed2004

Crystallization and preliminary crystallographic studies of a bifunctional restriction endonuclease Eco57I.

Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5'-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain. Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component. The best data for the complex with 25-mer DNA were collected to 4.2-A resolution at 100 K using synchrotron radiation. The crystals are orthorhombic, space group P2(1)2(1)2, with a=164.3, b=293.0, c=71.1 A, and contain two to four copies of the protein in the asymmetric unit.

Adenosine↗

PubMed2004

Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C.

Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.

Amyloid↗

PubMed2004

Evaluation of insulin sensitivity and beta-cell function indexes obtained from minimal model analysis of a meal tolerance test.

Modeling analysis of glucose, insulin, and C-peptide following a meal has been proposed as a means to estimate insulin sensitivity (S(i)) and beta-cell function from a single test. We compared the model-derived meal indexes with analogous indexes obtained from an intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp (HGC) in 17 nondiabetic subjects (14 men, 3 women, aged 50 +/- 2 years [mean +/- SE], BMI 25.0 +/- 0.7 kg/m(2)). S(i) estimated from the meal was correlated with S(i) estimated from the IVGTT and the HGC (r = 0.59 and 0.76, respectively; P < 0.01 for both) but was approximately 2.3 and 1.4 times higher (P < 0.05 for both). The meal-derived estimate of the beta-cell's response to a steady-state change in glucose (static secretion index) was correlated with the HGC second-phase insulin response (r = 0.69; P = 0.002), but the estimated rate-of-change component (dynamic secretion index) was not correlated with first-phase insulin release from either the HGC or IVGTT. Indexes of beta-cell function obtained from the meal were significantly higher than those obtained from the HGC. In conclusion, insulin sensitivity and beta-cell indexes derived from a meal are not analogous to those from the clamp or IVGTT. Further work is needed before these indexes can be routinely used in clinical and epidemiological studies.

Blood Glucose↗

PubMed2003

Modeling beta-cell insulin secretion--implications for closed-loop glucose homeostasis.

Glucose sensing and insulin delivery technology can potentially be linked to form a closed-loop insulin delivery system. Ideally, such a system would establish normal physiologic glucose profiles. To this end, a model of beta-cell secretion can potentially provide insight into the preferred structure of the insulin delivery algorithm. Two secretion models were evaluated for their ability to describe plasma insulin dynamics during hyperglycemic clamps (humans; n=7), and for their ability to establish and maintain fasting euglycemia under conditions simulated by the minimal model. The first beta-cell model (SD) characterized insulin secretion as a static component that had a delayed response to glucose, and a dynamic component that responded to the rate of increase of glucose. The second model (PID) described the response in terms of a proportional component without delay, an integral component that adjusted basal delivery in proportion to hyper/hypoglycemia, and a derivative component that responded to the rate of glucose change. Both models fit the beta-cell response during the clamp, and established fasting euglycemia under simulated closed-loop conditions; however, the SD model did not maintain euglycemia following simulated changes in insulin sensitivity or glucose appearance, whereas the PID model did. The PID model was more stable under the simulated closed-loop conditions. Both the SD and PID models described beta-cell secretion in response to a rapid increase in glucose. However, the PID model could maintain fasting euglycemia and was more stable under closed-loop conditions, and thus is more suited for such conditions.

Algorithms↗

PubMed2002

Crystallization and preliminary crystallographic studies of Streptococcus pyogenes cysteine protease precursor.

Streptococcal protease precursor, secreted by the human pathogen Streptococcus pyogenes, becomes activated to a cysteine protease. The precursor and the mature enzyme appear to contribute to S. pyogenes virulence. The precursor protein was crystallized in the form of very thin flexible flakes. X-ray diffraction data were collected to 3.15 A resolution at 100 K using synchrotron radiation. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 41.6, b = 136.0, c = 156.7 A, beta = 95.7 degrees, and contain four copies of the protein in the asymmetric unit.

Crystallization↗

PubMed2001

Crystallization and preliminary crystallographic studies of five crystal forms of Escherichia coli L-asparaginase II (Asp90Glu mutant).

L-Asparaginase II from Escherichia coli with an Asp90Glu mutation in the active site has been crystallized in five polymorphic forms. Crystals of all polymorphs suitable for X-ray diffraction experiments were obtained by the vapour-diffusion method. Crystals of form I belong to the monoclinic system (space group C2), have unit-cell parameters a = 73.1, b = 133.1, c = 62.6 A, beta = 108.8 degrees and diffract to 2.27 A resolution. Three of the crystal forms are orthorhombic, with unit-cell parameters a = 225.4, b = 128.0, c = 62.6 A (form II, P2(1)2(1)2), a = 59.9, b = 71.2, c = 130.6 A (form III, primitive cell) and a = 73.8, b = 122.1, c = 124.2 A (form IV, P2(1)2(1)2(1) or P2(1)2(1)2); the crystals diffract to 2.33, 3.5 and 1.7 A, respectively. Polymorph V is trigonal, space group P3(1)21, with unit-cell parameters a = 123.1, c = 83.8 A; the crystals diffract to 2.65 A resolution.

Asparaginase↗