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Aging slowly erodes skeletal muscle strength and mass, eventually leading to profound functional deficits and muscle atrophy. The molecular mechanisms of skeletal muscle aging are not well understood. To better understand mechanisms of muscle aging, we investigated the potential role of ATF4, a transcription regulatory protein that can rapidly promote skeletal muscle atrophy in young animals deprived of adequate nutrition or activity. To test the hypothesis that ATF4 may be involved in skeletal muscle aging, we studied fed and active muscle-specific ATF4 knockout mice (ATF4 mKO mice) at 6 months of age, when wild-type mice have achieved peak muscle mass and function, and at 22 months of age, when wild-type mice have begun to manifest age-related muscle atrophy and weakness. We found that 6-month-old ATF4 mKO mice develop normally and are phenotypically indistinguishable from 6-month-old littermate control mice. However, as ATF4 mKO mice become older, they exhibit significant protection from age-related declines in strength, muscle quality, exercise capacity, and muscle mass. Furthermore, ATF4 mKO muscles are protected from some of the transcriptional changes characteristic of normal muscle aging (repression of certain anabolic mRNAs and induction of certain senescence-associated mRNAs), and ATF4 mKO muscles exhibit altered turnover of several proteins with important roles in skeletal muscle structure and metabolism. Collectively, these data suggest ATF4 as an essential mediator of skeletal muscle aging and provide new insight into a degenerative process that impairs the health and quality of life of many older adults.

Skeletal muscle is critical for maintaining mobility, independence, and metabolic health in older adults. However, a common feature of aging is the progressive loss of skeletal muscle mass and function, which is often accompanied by mitochondrial impairments, oxidative stress, and insulin resistance. Exercise improves muscle strength, mitochondrial health, and cardiorespiratory fitness, but older adults often exhibit attenuated anabolic responses to acute exercise. Chronic inflammation associated with aging may contribute to this “anabolic resistance” and therapeutic interventions that target inflammation may improve exercise responsiveness. To this end, we conducted a randomized controlled trial to determine the effect of 6 months of dietary omega-3 polyunsaturated fatty acids (n3-PUFA) supplementation on skeletal muscle function (mass, strength), mitochondrial physiology (respiration, ATP production, ROS generation), and acute exercise responsiveness at the level of the muscle (fractional synthesis rate) and the whole-body (amino acid kinetics) in healthy older adults. When compared with a corn oil placebo (n = 33; 71.5 ± 4.8 years), older adults treated with 4 g/day n3-PUFA (n = 30; 71.4 ± 4.5 years) exhibited modest but significant increases in muscle strength (3.1 ± 14.7% increase in placebo vs. 7.5 ± 14.1% increase in n3-PUFA; p = 0.039). These improvements in muscle strength with n3-PUFA supplementation occurred in the absence of any effects on mitochondrial function and a minor attenuation of the acute response to exercise compared to placebo. Together, these data suggest modest benefits of dietary n3-PUFAs to muscle function in healthy older adults. Future studies may elucidate whether n3-PUFA supplementation improves the exercise response in elderly individuals with co-morbidities, such as chronic inflammatory disease or sarcopenia.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL-6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL-6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL-6 to evaluate the dose- and duration-dependent effects of IL-6 on salient aspects of mitochondrial biology and the role of canonical IL-6 signaling in muscle cells. Acute exposure of myotubes to IL-6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates (JO2 ) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL-6 was partly attenuated by MitoQ, a mitochondrial-targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL-6 exposure. In concert with effects on mitochondrial physiology, acute IL-6 exposure induced several mitochondrial adaptations, consistent with the stress-induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL-6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle.

Sarcopenia is linked with impaired adaptive responses to exercise in aging skeletal muscle. The unfolded protein response (UPR) is an important intramyocellular molecular response pathway that is activated by exercise. The influence of age on skeletal muscle adaptive UPR in response to exercise, and the relationship to other key exercise-responsive regulatory pathways is not well-understood. We evaluated age-related changes in transcriptional markers of UPR activation following a single bout of resistance exercise in 12 young (27 ± 5yrs) and 12 older (75 ± 5yrs) healthy men and women. At baseline, there were modest differences in expression of UPR-related genes in young and older adults. Following exercise, transcriptional markers of UPR pathway activation were attenuated in older adults compared to young based on specific salient UPR-related genes and gene set enrichment analysis. The coordination of post-exercise transcriptional patterns between the UPR pathway, p53/p21 axis of autophagy, and satellite cell differentiation were less evident in older compared to young adults. In conclusion, transcriptomic analysis revealed an age-related decline in the adaptive UPR transcriptional response following a single bout of exercise that could contribute to impaired exercise responsiveness with age.

Peptides presented by MHC molecules on the cell surface, or the immunopeptidome, play an important role in the adaptive arm of the immune response. Antigen processing for MHC class I molecules is a ubiquitous pathway present in all nucleated cells which generates and presents peptides of both self and non-self-origin. Peptides with post-translational modifications represent one category of peptides presented by MHC class I molecules. However, owing to the complexity of self-peptides presented by cells, the diversity of peptides with post-translational modifications is not well-studied. In this study, we carried out MHC Class I immunopeptidomics analysis of Loucy T-cell leukemia and A375 malignant melanoma cell line to characterize the diversity of post-translational modifications of MHC class I-bound peptides. Using high resolution mass spectrometry, we identified 25,761 MHC-bound peptides across both cell lines using Bolt and Sequest search engines. The enrichment method was highly specific as ~ 90% of the peptides were of typical length (8-12 amino acids long) and the motifs were expected based on previously reported motifs for MHC I alleles. Among the MHC-bound peptides, we identified phosphorylation as a major post-translational modification followed by deamidation. We observed site-specific localization of these post-translational modifications, at position P4 for phosphorylated peptides and position P3 for deamidated peptides. We identified a smaller number of peptides with acetylated and methylated lysine, possibly due to very low stoichiometric levels of these PTMs compared to phosphorylation and deamidation. Using PEAKS de novo sequencing algorithm, we identified spliced peptides that accounted for ~ 5-7% of MHC-bound peptides that were otherwise similar in their features as normal MHC-bound peptides. We validated the identity of several post-translationally modified peptides and spliced peptides through mass spectrometric analysis of synthetic peptides. Our study confirms post-translationally modified peptides to be present at low stoichiometric levels along with unusual spliced peptides through unbiased identification using high resolution mass spectrometry.The online version contains supplementary material available at 10.1007/s42485-021-00066-x.

Obesity is accompanied by numerous systemic and tissue-specific derangements, including systemic inflammation, insulin resistance, and mitochondrial abnormalities in skeletal muscle. Despite growing recognition that adipose tissue dysfunction plays a role in obesity-related disorders, the relationship between adipose tissue inflammation and other pathological features of obesity is not well-understood. We assessed macrophage populations and measured the expression of inflammatory cytokines in abdominal adipose tissue biopsies in 39 nondiabetic adults across a range of body mass indexes (BMI 20.5–45.8 kg/m(2)). Skeletal muscle biopsies were used to evaluate mitochondrial respiratory capacity, ATP production capacity, coupling, and reactive oxygen species production. Insulin sensitivity (S(I)) and β cell responsivity were determined from test meal postprandial glucose, insulin, c-peptide, and triglyceride kinetics. We examined the relationships between adipose tissue inflammatory markers, systemic inflammatory markers, S(I), and skeletal muscle mitochondrial physiology. BMI was associated with increased adipose tissue and systemic inflammation, reduced S(I), and reduced skeletal muscle mitochondrial oxidative capacity. Adipose-resident macrophage numbers were positively associated with circulating inflammatory markers, including tumor necrosis factor-α (TNFα) and C-reactive protein (CRP). Local adipose tissue inflammation and circulating concentrations of TNFα and CRP were negatively associated with S(I), and circulating concentrations of TNFα and CRP were also negatively associated with skeletal muscle oxidative capacity. These results demonstrate that obese humans exhibit increased adipose tissue inflammation concurrently with increased systemic inflammation, reduced insulin sensitivity, and reduced muscle oxidative capacity and suggest that adipose tissue and systemic inflammation may drive obesity-associated metabolic derangements. NEW AND NOTEWORTHY Adipose inflammation is proposed to be at the nexus of the systemic inflammation and metabolic derangements associated with obesity. The present study provides evidence to support adipose inflammation as a central feature of the pathophysiology of obesity. Adipose inflammation is associated with systemic and peripheral metabolic derangements, including increased systemic inflammation, reduced insulin sensitivity, and reduced skeletal muscle mitochondrial respiration.

Cancer cachexia is associated with muscle weakness and atrophy. We investigated whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), which has previously been shown to increase skeletal myoblast oxygen consumption rate, could reverse the deleterious effects of tumor cell conditioned medium on myoblast function. Conditioned medium from Lewis lung carcinoma (LLC1) cells inhibits oxygen consumption, increases mitochondrial fragmentation, inhibits pyruvate dehydrogenase activity, and enhances proteasomal activity in human skeletal muscle myoblasts. 1α,25(OH)2D3 reverses the tumor cell-mediated changes in mitochondrial oxygen consumption and proteasomal activity, without changing pyruvate dehydrogenase activity. 1α,25(OH)2D3 might be useful in treatment of weakness seen in association with CC.

Senescent cells accumulate in numerous tissues in several chronic conditions such as aging, obesity, and diabetes. These cells are in a state of irreversible cell-cycle arrest and secrete inflammatory cytokines, chemokines and other immune modulators that have paracrine effects on nearby tissues. Adipose tissue, in particular, harbors senescent cells, which have been linked with numerous chronic conditions and age-related comorbidities. Here we performed a series of in vitro experiments to determine the influence of senescent preadipocytes on key cell types found in vessel walls, including vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages (MQs), and adipose-derived stromal/stem cells (ASCs). Primary human preadipocytes were irradiated to trigger a senescence-like phenotype. VSMCs, ECs, MQs, and ASCs were exposed to conditioned media collected from irradiated preadipocytes or control preadipocytes. Additional experiments were performed where VSMCs, ECs, MQs, and ASCs were co-cultured with irradiated or control preadipocytes. The secretome of irradiated cells induced an inflammatory phenotype, decreased cell viability, disrupted proliferation and migration, and impaired metabolic function of these cell types in vitro. These maladaptive changes in response to senescent cell exposure provide early evidence in support of a hypothesis that senescent preadipocytes trigger phenotypic and functional changes in key cellular components of blood vessels that may contribute to vascular disease.

Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength.

The physiological importance of the intestinal plasma membrane calcium pump, isoform 1, (Pmca1, Atp2b1), in calcium absorption and homeostasis has not been previously demonstrated in vivo. Since global germ-line deletion of the Pmca1 in mice is associated with embryonic lethality, we selectively deleted the Pmca1 in intestinal absorptive cells. Mice with loxP sites flanking exon 2 of the Pmca1 gene (Pmca1(fl/fl)) were crossed with mice expressing Cre recombinase in the intestine under control of the villin promoter to give mice in which the Pmca1 had been deleted in the intestine (Pmca1(EKO) mice). Pmca1(EKO) mice were born at a reduced frequency and were small at the time of birth when compared to wild-type (Wt) littermates. At two months of age, Pmca1(EKO) mice fed a 0.81% calcium, 0.34% phosphorus, normal vitamin D diet had reduced whole body bone mineral density (P

Inactivating mutations of the SOST (sclerostin) gene are associated with overgrowth and sclerosis of the skeleton. To determine mechanisms by which increased amounts of calcium and phosphorus are accreted to enable enhanced bone mineralization in the absence of sclerostin, we measured concentrations of calciotropic and phosphaturic hormones, and urine and serum calcium and inorganic phosphorus in mice in which the sclerostin ( sost ) gene was replaced by the β-D-galactosidase ( lacZ ) gene in the germ line. Knockout (KO) ( sost −/− ) mice had increased bone mineral density and content, increased cortical and trabecular bone thickness, and greater net bone formation as a result of increased osteoblast and decreased osteoclast surfaces compared with wild-type (WT) mice. β-Galactosidase activity was detected in osteocytes of sost KO mice but was undetectable in WT mice. Eight-week-old, male sost KO mice had increased serum 1α,25-dihydroxyvitamin D, decreased 24,25-dihydroxyvitamin D, decreased intact fibroblast growth factor 23, and elevated inorganic phosphorus concentrations compared with age-matched WT mice. 25-Hydroxyvitamin D 1α-hydroxylase cytochrome P450 ( cyp27B1 ) mRNA was increased in kidneys of sost KO mice compared with WT mice. Treatment of cultured proximal tubule cells with mouse recombinant sclerostin decreased cyp27B1 mRNA transcripts. Urinary calcium and renal fractional excretion of calcium were decreased in sost KO mice compared with WT mice. Sost KO and WT mice had similar serum calcium and parathyroid hormone concentrations. The data show that sclerostin not only alters bone mineralization, but also influences mineral metabolism by altering concentrations of hormones that regulate mineral accretion.

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKO(OCN) mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKO(OCN) mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKO(OCN) mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKO(OCN) mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging.

To assess cellular mechanisms that could contribute to muscle weakness in the context of cancer cachexia, we evaluated the effect of conditioned medium (CM) from highly tumorigenic Lewis lung carcinoma (LLC1) cells, CM from less tumorigenic MLE12 cells and non-CM on human skeletal muscle mitochondrial morphology and function. We assessed mitochondrial oxygen consumption, morphology, enzyme activity and gene expression in human skeletal muscle cells in the presence of tumor conditioned medium or control medium. We examined the secretome of LLC1 cells for mediators of skeletal muscle changes. We demonstrate that LLC1 CM inhibits cellular O2 consumption rate (OCR) and increases the proton production rate in human skeletal muscle cells. Mitochondrial fragmentation and the expression of the fission mediator, FIS1, are increased, whereas the expression of fusion mediators, OPA1 and MFN2 are reduced by LLC1 CM. LLC1 CM reduces pyruvate dehydrogenase (PDH) activity thereby limiting the production of acetyl CoA. Reduced PDH activity is associated with reduced PDHA1 sub-unit expression. The amount inactive phospho-PDH is increased in association with reduced PDP2 expression and increased PDK4 and SIRT4 expression in human skeletal muscle cells treated with LLC1 CM. The expression of several signaling pathways is altered by LLC1 CM in human skeletal muscle cells. When compared to MLE12 cells, LLC1 cells show increased expression of mRNAs encoding several potential mediators of altered muscle function such as Gdf15, TGFβ-induced protein, IL11, Il27, Il33, Il34, and PTH-like protein. We show that the active vitamin D metabolite, 1α,25(OH)2D3, that we previously demonstrated increases myoblast mitochondrial oxygen consumption and PDH activity, mitigates LLC1 CM effects on hSkMC OCR, mitochondrial fragmentation and mediators thereof. Mitochondrial oxygen consumption, morphology and enzyme activity in human skeletal muscle cells are inhibited or altered by tumor cell conditioned medium, and are reversed by 1α,25(OH)2D3, the active metabolite of vitamin D. Citation Format: Zachary C. Ryan, Theodore A. Craig, Xuewei Wang, Philippe Delmotte, Jeffrey L. Salisbury, Ian R. Lanza, Gary C. Sieck, Rajiv Kumar. 1,25-Dihydroxyvitamin D3 mitigates lung cancer cell mediated mitochondrial dysfunction in human skeletal muscle [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5232.

The Calcium-Sensing Receptor (CaSR) is a G-protein-coupled receptor that regulates calcium homeostasis by altering parathyroid hormone release, and which binds divalent and trivalent cations, amino acids, polyamines, and polycationic ligands. To obtain information about the structural properties of the CaSR, we expressed milligram quantities of a pure, homogeneous, and functional fragment of the human CaSR extracellular domain (residues 20-535). The expressed and purified protein is folded and binds both neomycin and calcium. It forms dimers in the absence of reducing agents such as beta-mercaptoethanol. Thermal denaturation studies show it has enthalpy and entropy values of unfolding equal to DeltaH=-178+/-4 kJ/mol and DeltaS=-535+/-13 J/mol/K. The protein has significant secondary structure with alpha-helical, beta-sheet, beta-turns, and disordered content of 36.6+/-6.7%, 13.3+/-5.3%, 20.2+/-3.3%, and 29.4+/-4.0%, respectively. The described method for the expression and purification of CaSR should prove useful for further structural studies of this physiologically important protein.

Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered alpha-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an alpha-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.

Although many wood decay basidiomycetes secrete oxidative and hydrolytic enzymes that participate in lignocellulose biodedgradation, it is generally recognized now that these enzymes cannot penetrate sound wood, and that fungi must employ smaller agents to initiate decay. Reactive oxygen species (ROS) are likely candidates, and evidence is accumulating that some wood decay fungi produce these oxidants. For example, we have observed that, when they grow on wood, white rot and brown rot basidiomycetes degrade polyethylene glycol, a recalcitrant polymer that resists attack by species other than nonspecific, extracellular oxidants. By contrast, xylariaceous ascomycetes do not degrade polyethylene glycol, and therefore do not produce these oxidants when they degrade wood. The ROS that basidiomycetes employ remain unidentified in most cases, but the hydroxyl radical (·OH) is a likely participant, because there is a well-established pathway for its production via the Fenton reaction (H 2 O 2 + Fe 2+ + H + → H 2 O + Fe 3+ + ·OH). The two best documented fungal pathways for ·OH production involve the extracellular reduction of Fe 3+ and O 2 by cellobiose dehydrogenase or by secreted hydroquinones. Peroxyl (ROO·) and hydroperoxyl (·OOH) radicals, although they are weaker oxidants, may also be involved in wood decay because they are produced via reactions of ·OH and by some fungal enzymes.

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.

We investigated the influence of the osteocyte protein, sclerostin, on fracture healing by examining the dynamics and mechanisms of repair of single-cortex, stabilized femoral defects in sclerostin knockout (Sost(-/-); KO) and sclerostin wild-type (Sost(+/+); WT) mice. Fourteen days following generation of bone defects, Sost KO mice had significantly more bone in the healing defect than WT mice. The increase in regenerating bone was due to an increase in the thickness of trabecularized spicules, osteoblast numbers and surfaces within the defect. Enhanced healing of bone defects in Sost KO mice was associated with significantly more activated β-catenin expression than observed in WT mice. The findings were similar to those observed in Axin2(-/-) mice, in which β-catenin signaling is known to be enhanced to facilitate bone regeneration. Taken together, these data indicate that enhanced β-catenin signaling is present in Sost(-/-) mice that demonstrate accelerated healing of bone defects, suggesting that modulation of β-catenin signaling in bone could be used to promote fracture repair.

We analyzed the metal-binding properties of human centrin-2 (HsCen-2) and followed the changes in HsCen-2 structure upon metal-binding using micro-electrospray ionization mass spectrometry (muESI-MS). Apo-HsCen-2 is mostly monomeric. The ESI spectra of HsCen-2 show two charge-state distributions, representing two conformations of the protein. HsCen-2 binds four moles calcium/mol protein: one mol of calcium with high affinity, one additional mol of calcium with lower affinity, and two moles of calcium at low affinity sites. HsCen-2 binds four moles of magnesium/mol protein. The conformation giving the lower charge-state HsCen-2 by ESI, binds calcium and magnesium more readily than does the higher charge-state HsCen-2. Both conformations of HsCen-2 bind calcium more readily than magnesium. Calcium was more effective in displacing magnesium bound to HsCen-2 than vice versa. Binding of a peptide from a known binding partner, the xeroderma pigmentosum complementation group protein C (XPC), to apo-HsCen-2, occurs in the presence or the absence of calcium. Near and far-UV CD spectra of HsCen-2 show little difference with addition of calcium or magnesium. Minor changes in secondary structure are noted. Melting curves derived from temperature dependence of molar ellipticity at 222 nm for HsCen-2 show that calcium increases protein stability whereas magnesium does not. Delta 25 HsCen-2 behaves similarly to HsCen-2. We conclude that HsCen-2 binds calcium and magnesium and that calcium modulates HsCen-2 structure and function by increasing its stability without undergoing significant changes in secondary or tertiary structure.

Myosin VII (M7) plays a role in adhesion in both Dictyostelium and mammalian cells where it is a component of a complex of proteins that serve to link membrane receptors to the underlying actin cytoskeleton. The nature of this complex is not fully known, prompting a search for M7-binding proteins. Co-immunoprecipitation experiments reveal that Dictyostelium M7 (DdM7) interacts with talinA, an actin-binding protein with a known role in cell-substrate adhesion. No additional proteins are observed in the immunoprecipitate, indicating that the interaction is direct. The N-terminal region of the DdM7 tail that lies between the region of predicted coil and the first MyTH4 domain is found to harbor the talinA binding site. Localization experiments reveal that talinA does not serve as a membrane receptor for DdM7 and vice versa. These findings reveal that talinA is a major DdM7 binding partner and suggest that their interaction induces a conformational change in each that, in combination with membrane receptor binding, promotes the assembly of a high avidity receptor complex essential for adhesion of the cell to substrata.

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k cat / K m for the quinones is greater than 10 8 M −1 s −1 . The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.

The brown rot fungus Gloeophyllum trabeum uses an extracellular hydroquinone-quinone redox cycle to reduce Fe 3+ and produce H 2 O 2 . These reactions generate extracellular Fenton reagent, which enables G. trabeum to degrade a wide variety of organic compounds. We found that G. trabeum secreted two quinones, 2,5-dimethoxy-1,4-benzoquinone (2,5-DMBQ) and 4,5-dimethoxy-1,2-benzoquinone (4,5-DMBQ), that underwent iron-dependent redox cycling. Experiments that monitored the iron- and quinone-dependent cleavage of polyethylene glycol by G. trabeum showed that 2,5-DMBQ was more effective than 4,5-DMBQ in supporting extracellular Fenton chemistry. Two factors contributed to this result. First, G. trabeum reduced 2,5-DMBQ to 2,5-dimethoxyhydroquinone (2,5-DMHQ) much more rapidly than it reduced 4,5-DMBQ to 4,5-dimethoxycatechol (4,5-DMC). Second, although both hydroquinones reduced ferric oxalate complexes, the predominant form of Fe 3+ in G. trabeum cultures, the 2,5-DMHQ-dependent reaction reduced O 2 more rapidly than the 4,5-DMC-dependent reaction. Nevertheless, both hydroquinones probably contribute to the extracellular Fenton chemistry of G. trabeum , because 2,5-DMHQ by itself is an efficient reductant of 4,5-DMBQ.