William J. Cook

Structure of ubiquitin refined at 1.8 A resolution.

The crystal structure of human erythrocytic ubiquitin has been refined at 1.8 A resolution using a restrained least-squares procedure. The crystallographic R-factor for the final model is 0.176. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.5 degrees, respectively. A total of 58 water molecules per molecule of ubiquitin are included in the final model. The last four residues in the molecule appear to have partial occupancy or large thermal motion. The overall structure of ubiquitin is extremely compact and tightly hydrogen-bonded; approximately 87% of the polypeptide chain is involved in hydrogen-bonded secondary structure. Prominent secondary structural features include three and one-half turns of alpha-helix, a short piece of 3(10)-helix, a mixed beta-sheet that contains five strands, and seven reverse turns. There is a marked hydrophobic core formed between the beta-sheet and alpha-helix. The molecule features a number of unusual secondary structural features, including a parallel G1 beta-bulge, two reverse Asx turns, and a symmetrical hydrogen-bonding region that involves the two helices and two of the reverse turns.

Structure of calmodulin refined at 2.2 A resolution.

The crystal structure of mammalian calmodulin has been refined at 2.2 A (1 A = 0.1 nm) resolution using a restrained least-squares method. The final crystallographic R-factor, based on 6685 reflections in the range 2.2 A less than or equal to d less than or equal to 5.0 A with intensities exceeding 2.5 sigma, is 0.175. Bond lengths and bond angles in the molecule have root-mean-square deviations from ideal values of 0.016 A and 1.7 degrees, respectively. The refined model includes residues 5 to 147, four Ca2+ and 69 water molecules per molecule of calmodulin. The electron density for residues 1 to 4 and 148 is poorly defined, and they are not included in the model. The molecule is shaped somewhat like a dumbbell, with an overall length of 65 A; the two lobes are connected by a seven-turn alpha-helix. Prominent secondary structural features include seven alpha-helices, four Ca2+-binding loops, and two short, double-stranded antiparallel beta-sheets between pairs of adjacent Ca2+-binding loops. The four Ca2+-binding domains in calmodulin have a typical EF hand conformation (helix-loop-helix) and are similar to those described in other Ca2+-binding proteins. The X-ray structure determination of calmodulin shows a large hydrophobic cleft in each half of the molecule. These hydrophobic regions probably represent the sites of interaction with many of the pharmacological agents known to bind to calmodulin.

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 Å resolution

Ubiquitin C‐terminal hydrolases catalyze the removal of adducts from the C‐terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C‐terminal Hydrolase (UCH‐L3) by X‐ray crystallography at 1.8 å resolution. The structure is comprised of a central antiparallel β‐sheet flanked on both sides by α‐helices. The β‐sheet and one of the helices resemble the well‐known papain‐like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH‐L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH‐L3 differ, however, in strand and helix connectivity, which in the UCH‐L3 structure includes a disordered 20 residue loop (residues 147‐166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain‐like enzymes, we propose a model describing the binding of ubiquitin to UCH‐L3. The UCH‐L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9‐12 and 90‐94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non‐specific hydrolysis.

Peritransplant tolerance induction in macaques: early events reflecting the unique synergy between immunotoxin and deoxyspergualin.

BACKGROUND Day of transplant T cell depletion with anti-CD3 immunotoxin or F(Ab)2 immunotoxin induces stable tolerance to renal allografts in rhesus monkeys given 15-deoxyspergualin (DSG), a NF-kappaB inhibitor that suppresses proinflammatory cytokine (PC) production. Because PC and NF-kappaB are involved in dendritic cell (DC) maturation, we asked if impaired DC maturation and Th2-type cytokine deviation might be related to the synergistic effect of DSG in this novel model. METHODS Immunosuppression was initiated 4 hr before transplanting a major histocompatibility complex mismatched renal allograft. Some groups received a supplemental 5-day course of cyclosporine A or DSG or a 15-day course of DSG. Peripheral lymph nodes were sequentially examined for presence of mature DC. In vitro effects of DSG on PC-induced maturation of DC were also examined. RESULTS Allografts survived without rejection in 87% of recipients given immunotoxin or F(Ab)2 immunotoxin with DSG x 15 days, in 50% with DSG x 5 days, and 0% with cyclosporine A. The longest DSG survivors are >1000 days with normal graft function and tolerance validated, including acceptance of challenge second donor kidneys without treatment. DSG-treated recipients were unique in developing polarized Th2-type plasma cytokines. In DSG recipients, mature DC were significantly reduced in day +5 lymph node biopsies, with complete repopulation by 30 days. In vitro studies verified an inhibitory effect of DSG on DC maturation. CONCLUSIONS The study suggests DSG arrests DC maturation. The unusual synergy of immunotoxin and DSG apparently involves coincidental reduction in lymph node T cell mass and mature DC, a transient circumstance favoring development of stable tolerance.

Brief Communication: Glomerulonephritis in Patients with Hepatitis C Cirrhosis Undergoing Liver Transplantation

Context We do not know why some patients infected with hepatitis C virus (HCV) develop renal failure after liver transplantation. Contribution This case series describes 30 patients who had kidney biopsies during liver engraftment for HCV-induced cirrhosis. Twenty-five had immune-complex glomerulonephritis. Of these, 10 had normal serum creatinine levels, urinalysis results, and urinary protein excretion and none had blood or kidney cryoglobulins. Cautions Investigators did not study clinical outcomes after transplantation. Implications Clinically silent immune-complex glomerulonephritis occurs in patients with end-stage HCV-induced cirrhosis. We do not yet know whether it can lead to renal failure after liver transplantation. The Editors Chronic infection with hepatitis C virus (HCV) substantially impacts health care in the United States; currently, it affects approximately 2.7 million persons (1, 2). Accompanying extrahepatic syndromes may include glomerulonephritis (3, 4). Transplantation is a life-saving procedure for patients with end-stage cirrhosis; this disease accounts for 30% to 50% of the liver transplant surgeries performed annually in the United States. Compared with HCV-negative transplant recipients, HCV-positive recipients have an increased risk for renal failure after engraftment (5), perhaps because of glomerular renal disease. However, it is unclear whether such disease is present at engraftment or develops later. We performed the current study to determine whether HCV-infected patients have substantial renal injury at liver transplantation. Methods Patients From January 2004 through June 2005, 48 HCV antibodypositive adults underwent deceased-donor liver transplantation at the University of Alabama at Birmingham. We enrolled all 37 patients who provided written informed consent and met the following inclusion criteria: age older than 18 years; positive results for HCV antibody on second-generation enzyme-linked immunosorbent assay; circulating HCV RNA; and cirrhosis documented by history, liver biopsy, or abdominal imaging. Patients were excluded if they had previous organ transplantation, concomitant renal transplantation, ABO-incompatible liver, or seropositivity for hepatitis B virus or HIV. The Institutional Review Board for Human Use at the University of Alabama at Birmingham approved this study. Study Procedures Within 6 months of expected liver transplantation, medical history was recorded, physical examination was performed, and blood and urine samples were collected. Blood studies included serum creatinine and albumin, blood urea nitrogen, cryoglobulins (University of Alabama Hospital laboratory [6], assay repeated 9 days later), rheumatoid factor (Behring RapiTex RF slide latex agglutination, Newark, New Jersey), complement component 3 (C3) and complement component 4 (C4) (nephelometry assay, Beckman Array 360 Protein System, Beckman Instruments, Fullerton, California), 50% hemolytic complement (CH50) assay (University of Alabama Hospital laboratory) (7), HCV RNA by polymerase chain reaction (Cobas TaqMan HCV test, Roche Diagnostics, Branchburg, New Jersey), and HCV genotype (GeneAmp 9700 thermal cycler, Applied Biosystems, Foster City, California). Clean-catch urine samples were collected for all patients except the 2 with hepatorenal syndrome, who had indwelling bladder catheters. We defined elevated serum creatinine level as at least 124 mol/L (1.4 mg/dL) in men or at least 115 mol/L (1.3 mg/dL) in women, pathologic proteinuria as urine protein of at least 1+ on dipstick urinalysis or a urinary proteincreatinine ratio greater than 0.3, and hematuria as urine blood of at least 1+ on dipstick urinalysis or more than 2 erythrocytes per high-power field in a resuspended sediment. During liver transplantation, a kidney biopsy specimen was obtained with a Bard Max-Core Disposable Biopsy Instrument (Bard, Covington, Georgia) after reperfusion of the hepatic allograft if there was no clinical coagulopathy. Two-micron sections were processed for routine light microscopy. Direct immunofluorescence studies used polyclonal antibodies for IgA, IgG, IgM, and C3 and C1q components of complement. One-micron sections were used for ultrastructural studies. Role of the Funding Source Roche Pharmaceuticals, Inc., provided funding for this study. The authors independently designed, wrote, and conducted the study; collected and analyzed the data; and prepared and approved the manuscript. Results Patients Renal biopsy was not performed in 7 patients because of ongoing coagulopathy (n= 6) or inadequate tissue (n= 1); these patients were excluded from further analysis. Of the 20 men and 10 women in the analysis, 25 were white, 4 were African American, and 1 was Hispanic. Mean age at transplantation was 53 years (SD, 8); ages ranged from 41 to 73 years. Twelve patients had a history of hypertension, and 6 had diabetes mellitus. Thirteen patients had received interferon- therapy for at least 3 months, but none had resolution of viremia. Two participants with the hepatorenal syndrome at engraftment required continuous low-flow hemodialysis. Laboratory Data The mean interval between pretransplantation laboratory testing and surgery was 21 days (SD, 47), ranging from 0 to 192 days. Mean Model for End-stage Liver Disease score, including the exemption for hepatoma, was 25 (SD, 6); the range was 16 to 40. Thirteen patients had a normal renal laboratory profile (normal serum creatinine level, no pathologic proteinuria, and no hematuria) (Table 1). Mean serum creatinine level in 28 patients without the hepatorenal syndrome was 112 mol/L (SD, 46) (1.27 mg/dL [SD, 0.52]); values ranged from 44 to 265 mol/L (0.5 to 3.0 mg/dL). Mean HCV RNA viral load was 320000 IU/mL (SD, 440000); genotype was type 1 in 23 of 28 patients tested. Cryoglobulinemia was not detected in the 20 patients tested. Level of C3 was low in 18 of 24 patients, C4 level was low in 7 of 24 patients, and CH50 level was low in 18 of 23 patients. Results of tests for rheumatoid factor were positive in 10 of 24 patients. Table 1. Baseline Demographic and Clinical Characteristics and Renal Biopsy Findings of the 30 Patients, Grouped by Renal Biopsy Diagnosis Renal Biopsy With 1 exception, biopsy specimens contained 10 to 40 (mean, 22) glomeruli. Global glomerulosclerosis was relatively mild; 4 specimens showed more than 20% obsolete glomeruli. Three specimens contained a single segmental sclerotic lesion, and 1 specimen contained 2. Twenty-nine kidney biopsy specimens showed mesangial proliferation by light microscopy with immunofluorescence staining for immunoglobulins, complement, or both (Table 1). However, 4 of these specimens had no deposits by electron microscopy: One showed focal segmental glomerulosclerosis, and 3 were diagnosed as minor glomerular abnormalities in the absence of well-defined, electron-dense deposits. The 1 specimen that appeared normal on immunofluorescence and electron microscopy contained only 3 glomeruli. No specimen showed features of cryoglobulins. Twelve specimens were classified as membranoproliferative glomerulonephritis (MPGN) type 1 but did not show lobular accentuation and marked endocapillary hypercellularity, both of which are common in idiopathic MPGN type 1. Instead, mild to moderate mesangial hypercellularity was observed, with focal duplication of glomerular basement membranes. Another unusual feature was the pattern of immunofluorescence staining. Ten specimens showed staining for all reagents; 1 lacked staining for only IgA and another for only C3. Electron microscopy demonstrated mesangial interposition with subendothelial and mesangial deposits. Three specimens contained occasional subepithelial deposits. Thirteen other biopsy specimens showed an immune-complex glomerulonephritis of 2 patterns. Seven exhibited IgA nephropathy; none had mesangial interposition, but 4 had subendothelial deposits. Six other specimens demonstrated mesangial glomerulonephritis with proliferation of mesangial cells and variable increases in mesangial matrix without mesangial interposition. Immunofluorescence staining for IgA was less intense than that for IgG or IgM; electron microscopy confirmed mesangial deposits. Correlation of Renal Biopsy Findings with Clinical and Laboratory Data Age, ethnicity, and sex appeared similar among the patients grouped by histologic diagnosis. Frequency of diabetes mellitus, hypertension, previous alcohol use, hepatocellular carcinoma, HCV genotype, treatment with interferon-, and viral load did not differ substantially among groups. Levels of C3, C4, and CH50 tended to be lower in patients with MPGN type 1. Eighteen patients had no evidence of renal injury by urinary testing, including 15 with immune-complex glomerulonephritis; of these, 10 had a normal serum creatinine level and, thus, a normal renal laboratory profile (Figure). Patients with MPGN type 1 were most likely to have a clinical renal abnormality (Tables 1 and 2). Figure. Features of glomeruli in a kidney biopsy specimen from a 49-year-old white man with mesangial glomerulonephritis. A B C D E F Table 2. Pretransplantation Laboratory Characteristics of 25 Patients with Hepatitis C Infection and Immune-Complex Glomerulonephritis at Liver Transplantation Discussion An immune-complex glomerulonephritis, characterized by IgA deposits and some mesangial hypercellularity, was very common in patients with end-stage cirrhosis due to chronic HCV infection. Three types of disease were observed: MPGN type 1, IgA nephropathy, and mesangial glomerulonephritis. These distinctive patterns were not associated with any demographic variable tested. The immunoglobulin staining in MPGN type 1 differed from that in patients with idiopathic MPGN; IgA was much more frequent (8). Cryoglobulins, often found in HCV-infected patients with MPGN (9), were not detected, even in rheumatoid factorpositive patients with urinary abnormalities. Subendothelial depos

Three-dimensional structure of recombinant human granulocyte-macrophage colony-stimulating factor.

The crystal structure of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. There are two molecules in the crystallographic asymmetric unit, which are related by an approximate non-crystallographic 2-fold axis. The overall structure is highly compact and globular with a predominantly hydrophobic core. The main structural feature of rhGM-CSF is a four alpha-helix bundle, which represents approximately 42% of the structure. The helices are arranged in a left-handed antiparallel bundle with two overhand connections. Within the connections is a two-stranded antiparallel beta-sheet. The tertiary structure of rhGM-CSF has a topology similar to that of porcine growth factor and interferon-beta. Most of the proposed critical regions for receptor binding are located on a continuous surface at one end of the molecule that includes the C terminus.

Retraction: Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Scoring system for renal pathology in Fabry disease: report of the International Study Group of Fabry Nephropathy (ISGFN)

BACKGROUND In Fabry nephropathy, alpha-galactosidase deficiency leads to accumulation of glycosphingolipids in all kidney cell types, proteinuria and progressive loss of kidney function. METHODS An international working group of nephrologists from 11 Fabry centres identified adult Fabry patients, and pathologists scored histologic changes on renal biopsies. A standardized scoring system was developed with a modified Delphi technique assessing 59 Fabry nephropathy cases. Each case was scored independently of clinical information by at least three pathologists with an average final score reported. RESULTS We assessed 35 males (mean age 36.4 years) and 24 females (43.9 years) who mostly had clinically mild Fabry nephropathy. The average serum creatinine was 1.3 mg/dl (114.9 micromol/l); estimated glomerular filtration rate was 81.7 ml/min/1.73 m(2) and urine protein to creatinine ratio was 1.08 g/g (122.0 mg/mmol). Males had greater podocyte vacuolization on light microscopy (mean score) and glycosphingolipid inclusions on semi-thin sections than females. Males also had significantly more proximal tubule, peritubular capillary and vascular intimal inclusions. Arteriolar hyalinosis was similar, but females had significantly more arterial hyalinosis. Chronic kidney disease stage correlated with arterial and glomerular sclerosis scores. Significant changes, including segmental and global sclerosis, and interstitial fibrosis were seen even in patients with stage 1-2 chronic kidney disease with minimal proteinuria. CONCLUSIONS The development of a standardized scoring system of both disease-specific lesions, i.e. lipid deposition related, and general lesions of progression, i.e. fibrosis and sclerosis, showed a spectrum of histologic appearances even in early clinical stage of Fabry nephropathy. These findings support the role of kidney biopsy in the baseline evaluation of Fabry nephropathy, even with mild clinical disease. The scoring system will be useful for longitudinal assessment of prognosis and responses to therapy for Fabry nephropathy.

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2·0 Å resolution☆

The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.

Calcium-carbohydrate bridges composed of uncharged sugars. Structure of a hydrated calcium bromide complex of α-fucose

X-ray diffraction data were used to determine the crystal structure of a hydrated CaBr2 complex of alpha-fucose, a common terminal sugar of oligosaccharide chains on glycoproteins. Crystals of C6H12O5-CaBr2-3H2O are orthorhombic, space group P212121, with A equals 14.360(2), B equals 12.896(3), and C equals 8.043(1) A. Intensity data for 1442 independent reflections were measured with an automated diffractometer. A trial structure, obtained by the heavy-atom method, was refined by least-squares to R equals 0.052. Ca-2+ is chelated by a pair of hydroxyl groups from each of tow symmetry-related fucose molecules and is coordinated to three water molecules. Thus the structure consists of hydrated fucose-calcium-fucose bridges. The bridge geometry, which is dictated by the coordination requirements of Ca-2+, is like that of other calcium-carbohydrate complexes. Our results indicate that calcium-fucose interactions can provide an effective, sterospecific mechanism for cross-linking carbo hydrate chains. Similar calcium-carbohydrate bridges may be involved in a variety of Ca-2+-dependent agglutination and adhesion processes.