Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.     

Relation between Red Cell Distribution Width and Fibroblast Growth Factor 23 Cleaving in Patients with Chronic Kidney Disease and Heart Failure

Objective

In chronic kidney disease (CKD), both anemia and deregulated phosphate metabolism are common and predictive of adverse outcome. Previous studies suggest that iron status influences phosphate metabolism by modulating proteolytic cleavage of FGF23 into C-terminal fragments. Red cell distribution width (RDW) was recently identified as a strong prognostic determinant for cardiovascular morbidity and mortality, independently of iron status. We assessed whether RDW is associated with FGF23 cleaving in CKD patients with heart failure.

Materials and Methods

The associations between RDW and either intact FGF23 (iFGF23), C-terminal FGF23 (cFGF23, reflecting iFGF23 and C-terminal fragments together) and the iFGF23/cFGF23 ratio were analyzed in 52 patients with CKD (eGFR 34,9 ± 13.9 ml/min/1.73m²) and chronic heart failure (CHF). Associations between RDW and FGF23 forms were studied by linear regression analysis adjusted for parameters of renal function, iron metabolism, phosphate metabolism and inflammation.

Results

Median cFGF23 levels were 197.5 [110–408.5] RU/ml, median iFGF23 levels were 107.3 [65.1–162.2] pg/ml and median FGF23 ratio was 0.80 [0.37–0.86]. Mean RDW was 14.1 ± 1.2%. cFGF23 and RDW were associated (β = 1.63x10^-3, P <0.001), whereas iFGF23 and RDW were not (β = -1.38x10^-3, P = 0.336). The iFGF23/cFGF23 ratio was inversely associated with RDW. The difference between cFGF23 and iFGF23 (cFGF23- iFGF23) was positively associated with RDW (β = 1.74x10^-3, P< 0.001). The association between cFGF23 and RDW persisted upon multivariable linear regression analysis, adjusted for parameters of renal function, phosphate metabolism, iron metabolism and inflammation (β = 0.97x10^-3, P = 0.047).

Conclusion

RDW is associated with cFGF23 but not with iFGF23 levels in patients with CKD and CHF. This suggests a connection between RDW and FGF23 catabolism, independent of iron status and inflammation. Future studies are needed to unravel underlying mechanisms and whether these pertain to the link between RDW and outcome.     

Identification of Three Novel Genetic Variants in the Melanocortin‐3 Receptor of Obese Children

The melanocortin‐3 receptor (MC3R), a G‐protein‐coupled receptor expressed in the hypothalamus, is a key component of the leptin‐melanocortin pathway that regulates energy homeostasis. It is suggested that an MC3R defect leads to an increased feed efficiency, by which nutrients are partitioned preferentially into fat. In this study, we hypothesized that early‐onset obesity could be induced by mutations in MC3R. To investigate this hypothesis, we screened the entire coding region of the MC3R gene for mutations in obese subjects. A total of 404 overweight and obese children and adolescents, 86 severely obese adults (BMI ≥40 kg/m²), and 150 normal‐weight control adults were included. Besides three synonymous coding variations in the MC3R gene (S69S, L95L, I226I), we were able to identify three novel heterozygous, nonsynonymous, coding mutations (N128S, V211I, L299V) in three unrelated obese children. None of these mutations were found in any of the control subjects. Functional studies assessing localization and signaling properties of the mutant receptors provided proof for impaired function of the L299V mutated receptor, whereas no conclusive evidence for functional impairment of the N128S and V211I mutated receptors could be established. First, these results provide supporting evidence for a role of the MC3R gene in the pathogenesis of obesity in a small subset of patients. Second, they show that caution is called for the interpretation of newly discovered mutations in MC3R.     

Analysis of genetic variations in the resistin gene shows no associations with obesity in women

Resistin is thought to be involved in the development of obesity and insulin resistance. Polymorphisms in the gene encoding resistin could contribute to this link, but different studies have yielded contradictory results. In this study, we investigated whether polymorphisms in resistin are involved in the development of obesity in a Belgian female population. We selected three single-nucleotide polymorphisms (SNPs; rs1862513, rs3745367, and rs3745369) and compared their genotype and allele frequencies between female obese patients (N = 541) and control individuals (N = 235). This analysis showed association with neither obesity for any of the variants nor with the haplotypes of these SNPs. Furthermore, we also investigated whether these variants have an influence on BMI. After Kruskal-Wallis analysis, we found that there was no difference in BMI between the genotypes for all variants. Together, these results suggest that these variants in resistin are not associated with obesity in the female population.     

Structure of a bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola: crystallographic evidence for a trimer

Crystals of the bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola, previously described by J. M. Olson et al. (1969), have been re-examined by X-ray diffraction. The space group is P6₃, as reported in the earlier work, but revised cell dimensions, $\text{a = b = 112.4 ± 0.4}\text{A}\text{?}\text{, c = 98.4 ± 0.4}\text{A}\text{?}$, were obtained, leading to a unit cell volume one third of that reported previously. Correction of this error leads to the conclusion that the bacteriochlorophyll-protein complex must be a trimer consisting of three identical subunits arranged about a crystallographic symmetry axis. Also a new trigonal crystal form of the bacteriochlorophyll-protein has been obtained, and is consistent only with a molecule composed of three identical or near-identical subunits. Models of the molecular packing for both crystal forms are presented.The molecular weight of the bacteriochlorophyll-protein complex, determined from crystal density measurements, is (1.53 ± 0.23) × 10⁵, and the overall molecular dimensions are about 55 Å along the trimer axis, and 83 Å at right angles to this. There are probably seven bacteriochlorophyll molecules in each subunit.     

Crystallographic analysis of the three-dimensional structure of baboon alpha-lactalbumin at low resolution. Homology with lysozyme.

Previous investigations [Jones, Brown, von Glos & Gaunt (1985) Exp. Cell Res. 156, 31-44] have demonstrated the appearance of a new antigenic determinant (recognized by monoclonal antibody 2D6) on the plasma membrane of rat spermatozoa during post-testicular maturation in the epididymis. Identification of the 2D6 antigen on Western blots from one-dimensional SDS/polyacrylamide gels revealed that it co-migrated with a membrane protein (designated Mr 23,000 antigen) present on testicular and immature germ cells, suggesting that one antigen might be a modified version of the other. In the present work, however, we demonstrate that, although they have similar Mr and are present in soluble and membrane-bound forms, the 2D6 and Mr 23,000 antigens are biochemically and immunologically distinct molecules. The properties of the antigens are described and compared. The Mr 23,000 antigen is present on both testicular and cauda epididymidal spermatozoa, has a pI of 6.1, contains no detectable carbohydrate, is not tissue-specific and is degraded by V8 protease. By contrast, the 2D6 antigen is glycosylated, has a broad pI from 4.5 to 6.1, is tissue- and species-specific and is resistant to digestion with V8 protease. Its role in sperm-egg recognition is discussed.     

Atomic coordinates for the chlorophyll core of a bacteriochlorophyll a-protein from green photosynthetic bacteria

Preliminary atomic coordinates are presented for the seven bacteriochlorophylls constituting the core of one subunit of the trimeric water soluble bacteriochlorophyll $\text{a}$-protein from the green photosynthetic bacterium $\text{Prosthecochloris aestuarii}$, strain 2K, formerly identified as $\text{Chlorobium limicola}$, strain 2K. The coordinates were derived from a 2.8 Å resolution electron density map based on four isomorphous heavy-atom derivatives, and adjusted to have stereochemically acceptable bond lengths and angles.     

Purified, Soluble Recombinant Mouse Hepatitis Virus Receptor, Bgp1b, and Bgp2 Murine Coronavirus Receptors Differ in Mouse Hepatitis Virus Binding and Neutralizing Activities

Mouse hepatitis virus receptor (MHVR) is a murine biliary glycoprotein (Bgp1^a). Purified, soluble MHVR expressed from a recombinant vaccinia virus neutralized the infectivity of the A59 strain of mouse hepatitis virus (MHV-A59) in a concentration-dependent manner. Several anchored murine Bgps in addition to MHVR can also function as MHV-A59 receptors when expressed at high levels in nonmurine cells. To investigate the interactions of these alternative MHVR glycoproteins with MHV, we expressed and purified to apparent homogeneity the extracellular domains of several murine Bgps as soluble, six-histidine-tagged glycoproteins, using a baculovirus expression system. These include MHVR isoforms containing four or two extracellular domains and the corresponding Bgp1^b glycoproteins from MHV-resistant SJL/J mice, as well as Bgp2 and truncation mutants of MHVR and Bgp1^b comprised of the first two immunoglobulin-like domains. The soluble four-domain MHVR glycoprotein (sMHVR[1-4]) had fourfold more MHV-A59 neutralizing activity than the corresponding soluble Bgp1^b (sBgp1^b) glycoprotein and at least 1,000-fold more neutralizing activity than sBgp2. Although virus binds to the N-terminal domain (domain 1), soluble truncation mutants of MHVR and Bgp1^b containing only domains 1 and 2 bound virus poorly and had 10- and 300-fold less MHV-A59 neutralizing activity than the corresponding four-domain glycoproteins. In contrast, the soluble MHVR glycoprotein containing domains 1 and 4 (sMHVR[1,4]) had as much neutralizing activity as the four-domain glycoprotein, sMHVR[1-4]. Thus, the virus neutralizing activity of MHVR domain 1 appears to be enhanced by domain 4. The sBgp1^b[1-4] glycoprotein had 500-fold less neutralizing activity for MHV-JHM than for MHV-A59. Thus, MHV strains with differences in S-glycoprotein sequence, tissue tropism, and virulence can differ in the ability to utilize the various murine Bgps as receptors.     

Crystallization of cow α-lactalbumin

Three new crystal forms of cow α-lactalbumin are described. A trigonal form in space group P3₁21 or P3₂21 has unit cell dimensions: $\text{a = b = 57.4}\text{A}\text{?}\text{, c = 75.0}\text{A}\text{?}$. A hexagonal form in space group P622 has unit cell dimensions: $\text{a = b = 94.0}\text{A}\text{?}\text{, c = 67.1}\text{A}\text{?}$. A second trigonal form grown in the presence of calcium ions belongs to space group P321 with unit cell dimensions: $\text{a = b = 93.7}\text{A}\text{?}\text{, c = 66.9}\text{A}\text{?}$. The significance of these new crystal forms to the structure determination of cow α-laetalbumin is discussed.