Search

An aged immune system undergoes substantial changes where myelopoiesis dominates within the bone marrow. Monocytic-MDSCs (M-MDSCs) have been found to play an important role in osteoclastogenesis and bone resorption. In this study, we sought to provide a more comprehensive understanding of the osteoclastogenic potential of bone marrow M-MDSCs during normal aging through transcriptomic and metabolic changes. Using young mature and aged mice, detailed immunophenotypic analyses of myeloid cells revealed that the M-MDSCs were not increased in bone marrow, however M-MDSCS were significantly expanded in peripheral tissues. Although aged mice exhibited a similar number of M-MDSCs in bone marrow, these M-MDSCs had significantly higher osteoclastogenic potential and greater demineralization activity. Intriguingly, osteoclast progenitors from aged bone marrow M-MDSCs exhibited greater mitochondrial respiration rate and glucose metabolism. Further, transcriptomic analyses revealed the upregulation of mitochondrial oxidative phosphorylation and glucose metabolism genes. Interestingly, there was 8-fold increase in Cd38 mRNA gene expression, consistent with the Mouse Aging Cell Atlas transcriptomic database, and confirmed by qRT-PCR. CD38 regulates NAD + availability, and 78c, a small molecule inhibitor of CD38, reduced the mitochondrial oxygen consumption rate and glucose metabolism and inhibited the osteoclastogenic potential of aged mice bone marrow-derived M-MDSCs. These results indicate that the age-related increase in Cd38 expression in M-MDSCs bias the transcriptome of M-MDSCs towards osteoclastogenesis. This enhanced understanding of the mechanistic underpinnings of M-MDSCs and their osteoclastogenesis during aging could lead to new therapeutic approaches for age-related bone loss and promote healthy aging., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Introduction: Frailty often manifests as an increased vulnerability to adverse outcomes, and detecting frailty is useful for informed healthcare decisions. Veterans are at higher risk for developing frailty and at younger ages. The goal of this study was to investigate approaches in Veterans that can better inform the physiologic underpinnings of frailty, including maximal oxygen uptake (VO2max), 6-min walk, muscle strength, and inflammatory biomarkers., Methods: Participants (N = 42) were recruited from the Buffalo VA Medical Center. Inclusion criteria: ages 60-85, male or female, any race, and not having significant comorbidities or cognitive impairment. Outcome measures included: the Fried frailty phenotype, the short physical performance battery (SPPB), quality of life (QOL) using the Q-LES-Q-SF, and the following physiologic assessments: VO2max assessment on an upright stationary bicycle, 6-min walk, and arm and leg strength. Additionally, inflammatory biomarkers (C-reactive protein, IL-6, IL-10, interferon-γ, and TNF-α) were measured using ELLA single and multiplex ELISA., Results: Participants: 70.3 ± 7.4 years of age: 34 males and 8 females, BMI = 30.7 ± 5.4 kg/m 2 , 26 white and 16 African American. A total of 18 (42.8%) were non-frail, 20 (47.6%) were pre-frail, and 4 (9.5%) were frail. VO2max negatively correlated with Fried frailty scores (r = -0.40, p = 0.03, N = 30), and positively correlated with SPPB scores (r = 0.50, p = 0.005), and QOL (r = 0.40, p = 0.03). The 6-min walk test also significantly correlated with VO2max (r = 0.57, p = 0.001, N = 42) and SPPB (r = 0.55, p = 0.0006), but did not quite reach a significant association with frailty (r = -0.28, p = 0.07). Arm strength negatively correlated with frailty (r = -0.47, p = 0.02, N = 26), but not other parameters. Inflammatory profiles did not differ between non-frail and pre-frail/frail participants., Conclusion: Objectively measured cardiorespiratory fitness was associated with important functional outcomes including physical performance, QOL, and frailty in this group of older Veterans. Furthermore, the 6-min walk test correlated with VO2max and SPPB, but more validation is necessary to confirm sensitivity for frailty. Arm strength may also be an important indicator of frailty, however the relationship to other indicators of physical performance is unclear., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Seldeen, Rahman, Redae, Satchidanand, Mador, Ma, Soparkar, Lima, Ezeilo and Troen.)

Mitochondrial dysfunction is a hallmark of cellular senescence, with the loss of mitochondrial function identified as a potential causal factor contributing to senescence-associated decline in cellular functions. Our recent findings revealed that ectopic expression of the pluripotency transcription factor NANOG rejuvenates dysfunctional mitochondria of senescent cells by rewiring metabolic pathways. In this study, we report that NANOG restores the expression of key enzymes, PYCR1 and PYCR2, in the proline biosynthesis pathway. Additionally, senescent mesenchymal stem cells manifest severe mitochondrial respiratory impairment, which is alleviated through proline supplementation. Proline induces mitophagy by activating AMP-activated protein kinase α and upregulating Parkin expression, enhancing mitochondrial clearance and ultimately restoring cell metabolism. Notably, proline treatment also mitigates several aging hallmarks, including DNA damage, senescence-associated β-galactosidase, inflammatory cytokine expressions, and impaired myogenic differentiation capacity. Overall, this study highlights the role of proline in mitophagy and its potential in reversing senescence-associated mitochondrial dysfunction and aging hallmarks., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, in vitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Osteoclastic bone resorption markedly increases with aging, leading to osteoporosis characterized by weak and fragile bones. Mice exhibit greater bone resorption and poor bone mass when Sirt1 is removed from their osteoclasts. Here we investigated the ex vivo impacts of putative Sirt1 activators, Resveratrol (RSV), SRT2183, and SRT1720, on osteoclast formation and activity in primary mouse bone marrow cells (BMCs) derived from wild-type (WT) and osteoclast specific Sirt1 knockout (OC-Sirt1KO) mice and in the RAW264.7 mouse macrophage cell line., Results: We found that SRT2183 and SRT1720 inhibit the formation of osteoclasts and actin belts in BMCs and RAW264.7 cells, whereas RSV does not. We also observed that the OC-Sirt1KO mice exhibited less bone mineral density, and the BMCs harvested from these mice yielded more osteoclasts than BMCs harvested from littermate controls. Interestingly, both SRT2183 and SRT1720 reduced osteoclast and actin belt formation in BMCs from OC-Sirt1KO mice. SRT2183 and SRT1720 also significantly disrupted actin belts of mature osteoclasts generated from BMCs of WT mice, within 3 and 6 hours of administration, respectively. Furthermore, these compounds inhibited the resorption activity of mature osteoclasts, while RSV did not., Conclusion: Our findings suggest SRT2183 and SRT1720 impede bone resorption by disrupting actin belts of mature osteoclasts, inhibit actin belt formation, and inhibit osteoclastogenesis even in the absence of Sirt1. Thus, the mechanism of action of these compounds appears to extend beyond Sirt1 activation and possibly pave the way for potential new therapies in alleviating osteoporosis associated bone loss., Competing Interests: COMPETING INTERESTS All authors have no conflicts of interest.

Mitochondrial dysfunction, a hallmark of aging, has been associated with the onset of aging phenotypes and age-related diseases. Here, we report that impaired mitochondrial function is associated with increased glutamine catabolism in senescent human mesenchymal stem cells (MSCs) and myofibroblasts derived from patients suffering from Hutchinson-Gilford progeria syndrome. Increased glutaminase (GLS1) activity accompanied by loss of urea transporter SLC14A1 induces urea accumulation, mitochondrial dysfunction, and DNA damage. Conversely, blocking GLS1 activity restores mitochondrial function and leads to amelioration of aging hallmarks. Interestingly, GLS1 expression is regulated through the JNK pathway, as demonstrated by chemical and genetic inhibition. In agreement with our in vitro findings, tissues isolated from aged or progeria mice display increased urea accumulation and GLS1 activity, concomitant with declined mitochondrial function. Inhibition of glutaminolysis in progeria mice improves mitochondrial respiratory chain activity, suggesting that targeting glutaminolysis may be a promising strategy for restoring age-associated loss of mitochondrial function., Competing Interests: Declaration of interests The authors declare that they do not have financial or nonfinancial competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aging results in enhanced myelopoiesis, which is associated with an increased prevalence of myeloid leukemias and the production of myeloid-derived suppressor cells (MDSCs). Tristetraprolin (TTP) is an RNA binding protein that regulates immune-related cytokines and chemokines by destabilizing target mRNAs. As TTP expression is known to decrease with age in myeloid cells, we used TTP-deficient (TTPKO) mice to model aged mice to study TTP regulation in age-related myelopoiesis. Both TTPKO and myeloid-specific TTPKO (cTTPKO) mice had significant increases in both MDSC subpopulations M-MDSCs (CD11b + Ly6C hi Ly6G - ) and PMN-MDSCs (CD11b + Ly6C lo Ly6G + ), as well as macrophages (CD11b + F4/80 + ) in the spleen and mesenteric lymph nodes; however, no quantitative changes in MDSCs were observed in the bone marrow. In contrast, gain-of-function TTP knock-in (TTPKI) mice had no change in MDSCs compared with control mice. Within the bone marrow, total granulocyte-monocyte progenitors (GMPs) and monocyte progenitors (MPs), direct antecedents of M-MDSCs, were significantly increased in both cTTPKO and TTPKO mice, but granulocyte progenitors (GPs) were significantly increased only in TTPKO mice. Transcriptomic analysis of the bone marrow myeloid cell populations revealed that the expression of CC chemokine receptor 2 (CCR2), which plays a key role in monocyte mobilization to inflammatory sites, was dramatically increased in both cTTPKO and TTPKO mice. Concurrently, the concentration of CC chemokine ligand 2 (CCL2), a major ligand of CCR2, was high in the serum of cTTPKO and TTPKO mice, suggesting that TTP impacts the mobilization of M-MDSCs from the bone marrow to inflammatory sites during aging via regulation of the CCR2-CCL2 axis. Collectively, these studies demonstrate a previously unrecognized role for TTP in regulating age-associated myelopoiesis through the expansion of specific myeloid progenitors and M-MDSCs and their recruitment to sites of injury, inflammation, or other pathologic perturbations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kwack, Zhang, Kramer, Thiyagarajan, Lamb, Arao, Bard, Seldeen, Troen, Blackshear, Abrams and Kirkwood.)

Objectives: Nicotinamide adenine dinucleotide (NAD + ), an essential cofactor for mitochondrial function, declines with aging, which may lead to impaired physical performance. Nicotinamide riboside (NR), a NAD + precursor, restores cellular NAD + levels. The aim of this study was to examine the effects of short-term NR supplementation on physical performance in middle-aged mice and the effects on mouse and human muscle stem cells., Methods: We treated 15-mo-old male C57BL/6J mice with NR at 300 mg·kg·d -1 (NR3), 600 mg·kg·d -1 (NR6), or placebo (PLB), n = 8 per group, and assessed changes in physical performance, muscle histology, and NAD + content after 4 wk of treatment., Results: NR increased total NAD + in muscle tissue (NR3 P = 0.01; NR6 P = 0.004, both versus PLB), enhanced treadmill endurance and open-field activity, and prevented decline in grip strength. Histologic analysis revealed NR-treated mice exhibited enlarged slow-twitch fibers (NR6 versus PLB P = 0.014; NR3 P = 0.16) and a trend toward more slow fibers (NR3 P = 0.14; NR6 P = 0.22). We next carried out experiments to characterize NR effects on mitochondrial activity and cellular energetics in vitro. We observed that NR boosted basal and maximal cellular aerobic and anaerobic respiration in both mouse and human myoblasts and human myotubes. Additionally, NR treatment improved the differentiating capacity of myoblasts and increased myotube size and fusion index upon stimulation of these progenitors to form multinucleated myotubes., Conclusion: These findings support a role for NR in improving cellular energetics and functional capacity in mice, which support the translation of this work into clinical settings as a strategy for improving and/or maintaining health span during aging., (Published by Elsevier Inc.)

Frailty is a condition marked by greater susceptibility to adverse outcomes, including disability and mortality, which affects up to 50% of those 80 years of age and older. Concurrently, serum vitamin D insufficiency and deficiency, for which as many as 70% of older adults may be at risk, potentially play an important role in frailty onset and progression. Large population driven studies have uncovered associations between low serum vitamin D levels and higher incidence of frailty. However, attempts to apply vitamin D therapeutically to treat and/or prevent frailty have not yielded consistent support for benefits. Given the complexity and inconsistency arising from human studies involving vitamin D, our research group has recently published on animal models of vitamin D insufficiency. Combining our model with the emerging development of animal frailty assessment, we identified that higher than standard levels of vitamin D supplementation may delay frailty in mice. In this viewpoint article, we will discuss current knowledge regarding the importance of vitamin D in frailty progression, the emerging significance of animal models in addressing these relationships, and the future for pre-clinical and clinical research., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

Low 25-OH serum vitamin D (VitD) is pervasive in older adults and linked to functional decline and progression of frailty. We have previously shown that chronic VitD insufficiency in "middle-aged" mice results in impaired anaerobic exercise capacity, decreased lean mass, and increased adiposity. Here, we examine if VitD insufficiency results in similar deficits and greater frailty progression in old-aged (24 to 28 months of age) mice. Similar to what we report in younger mice, older mice exhibit a rapid and sustained response in serum 25-OH VitD levels to differential supplementation, including insufficient (125 IU/kg chow), sufficient (1000 IU/kg chow), and hypersufficient (8000 IU/kg chow) groups. During the 4-month time course, mice were assessed for body composition (DEXA), physical performance, and frailty using a Fried physical phenotype-based assessment tool. The 125 IU mice exhibited worse grip strength ( p = 0.002) and inverted grip hang time ( p = 0.003) at endpoint and the 8000 IU mice transiently displayed greater rotarod performance after 3 months ( p = 0.012), yet other aspects including treadmill performance and gait speed were unaffected. However, 125 and 1000 IU mice exhibited greater frailty compared to baseline ( p = 0.001 and p = 0.038, respectively), whereas 8000 IU mice did not ( p = 0.341). These data indicate targeting higher serum 25-OH vitamin D levels may attenuate frailty progression during aging.

Complement (C) system is a double edge sword acting as the first line of defense on the one hand and causing aggravation of disease on the other. C activation when unregulated affects different organs including muscle regeneration. However, the effect of factor H (FH), a critical regulator of the alternative C pathway in muscle remains to be studied. FH deficiency results in excessive C activation and generates proinflammatory fragments C5a and C3a as byproducts. C3a and C5a signal through their respective receptors, C5aR and C3aR. In this study, we investigated the role of FH and downstream C5a/C5aR signaling in muscle architecture and function. Using the FH knockout (fh-/-) and fh-/-/C5aR-/double knockout mice we explored the role of C, specifically the alternative C pathway in muscle dysfunction. Substantial C3 and C9 deposits occur along the walls of the fh-/- muscle fibers indicative of unrestricted C activation. Physical performance assessments of the fh-/- mice show reduced grip endurance (76 %), grip strength (14 %) and rotarod balance (36 %) compared to controls. Histological analysis revealed a shift in muscle fiber populations indicated by an increase in glycolytic MHC IIB fibers and reduction in oxidative MHC IIA fibers. Consistent with this finding, mitochondrial DNA (mtDNA) and citrate synthase (CS) expression were both reduced indicating possible reduction in mitochondrial biomass. In addition, our results showed a significant increase in TGFβ expression and altered TGFβ localization in this setting. The architecture of cytoskeletal proteins actin and vimentin in the fh-/- muscle was changed that could lead to contractile weakness and loss of skeletal muscle elasticity. The muscle pathology in fh-/- mice was reduced in fh-/-/C5aR-/- double knockout (DKO) mice, highlighting partial C5aR dependence. Our results for the first time demonstrate an important role of FH in physical performance and skeletal muscle health., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

This report summarizes the presentations and recommendations of the eleventh annual American Geriatrics Society and National Institute on Aging research conference, "Osteoporosis and Soft Tissue (Muscle/Fat) Disorders," on March 11-12, 2019, in Bethesda, Maryland. Falls, fractures, and sarcopenia have a major impact on health in older adults, and they are interconnected by known risk factors. The link between osteoporosis, which is common in older adults, and the risk of falls is well known. Sarcopenia, the age-related decline in skeletal muscle mass and function, is also associated with an increased risk of falls and fractures because it reduces strength and leads to functional limitations. In addition to increasing the risk of falls, sarcopenia and osteoporosis can lead to frailty, reduced quality of life, morbidity, and mortality. The conference highlighted the impact of bone and soft tissue disorders on quality of life, morbidity, and mortality in older adults. Presenters described factors that contribute to these disorders; health disparities experienced by various subpopulations; and promising biological, pharmacologic, and behavioral interventions to prevent or treat these disorders. The workshop identified many research gaps and questions along with research recommendations that have the potential to enhance the prospect of healthy aging and improved quality of life for older adults. J Am Geriatr Soc 68:31-38, 2019., (© 2019 The American Geriatrics Society.)

Cathepsin K (CTSK) is a secreted protease that plays an essential role in osteoclastic bone resorption and osteoporotic bone loss. We have previously shown that activator protein 1 (AP-1) stimulates CTSK promoter activity and that proximal nuclear factor of activated T cells cytoplasmic 1 (NFATc1)-binding sites play a major role in the stimulation of CTSK gene expression by receptor activator of NFκB ligand (RANKL). In the present study, we have extended these observations and further dissected the effects of transcription factors involved in the regulation of CTSK gene expression. Our aim was to investigate the cooperative interplay among transcription factors AP-1, microphthalmia-associated transcription factor (Mitf), and NFATc1, and the consequent regulatory effects on CTSK transcription. Experiments were carried out in RAW 264.7 cells, which can be readily differentiated to osteoclasts upon RANKL stimulation. Our data show that AP-1, Mitf, and NFATc1 are capable of independently stimulating CTSK promoter activity. A combination of any two factors further enhances CTSK promoter activity, with the combination of AP-1 (c-fos/c-jun) and NFATc1 inducing the largest increase. We further identify a synergistic effect when all three factors cooperate intimately at the proximal promoter region, yielding maximal transcriptional upregulation of the CTSK promoter. RANKL induces temporal localization of AP-1 and NFATc1 to the CTSK promoter. These results suggest that the interaction of multiple transcription factors mediate a maximal response to RANKL-induced CTSK gene expression., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Frailty syndrome increases the risk for disability and mortality, and is a major health concern amidst the geriatric shift in the population. High intensity interval training (HIIT), which couples bursts of vigorous activity interspersed with active recovery intervals, shows promise for the treatment of frailty. Here we compare and contrast five Fried physical phenotype and one deficit accumulation based mouse frailty assessment tools for identifying the impacts of HIIT on frailty and predicting functional capacity, underlying pathology, and survival in aged female mice. Our data reveal a 10-minute HIIT regimen administered 3-days-a-week for 8-weeks increased treadmill endurance, gait speed and maintained grip strength. One frailty tool identified a benefit of HIIT for frailty, but many were trending suggesting HIIT was beneficial for physical performance in these mice, but the 8-week timeframe may have been insufficient to induce frailty benefits. Finally, most frailty tools distinguished between surviving or non-surviving mice, whereas half correlated with functional capacity measured by nest building ability, and none correlated with underlying pathology. In summary, this study supports the ongoing development of mouse assessment tools as useful instruments for frailty research., (Copyright © 2019 Elsevier B.V. All rights reserved.)

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

Complement system is an important arm of the immune system that promotes inflammation. Complement Factor H (FH) is a critical regulator of the alternative complement pathway. Its absence causes pathology in different organs resulting in diseases such as age related macular degeneration and dense deposit disease. Recent studies suggest that the complement system plays a role in bone development and homeostasis. To determine the role of FH in bone architecture, we studied the FH knockout (fh-/-) mice. 3D reconstructions of femur from 16 week old fh-/- mice reveal significant changes, such as decreased BV/TV (4.5%, p < 0.02), trabecular number (22%, p < 0.01), tissue mineral density (16%, p < 0.04), and increased marrow area (16% p < 0.01), compared to their wild type (WT) counterparts. Kidney function and histology remained normal indicating that bone changes occurred prior to kidney dysfunction. Next we examined cultured osteoblasts and osteoclasts isolated from bone marrow. FH is expressed ubiquitously in the osteoblasts and in the cytoplasm of osteoclasts. The changes caused by absence of FH include: increase in number of osteoblasts (362%) and osteoclasts (342%), increase in RNA (180%) and protein expression of cathepsin K and increased osteoclast function (pit formation, 233%). Actin rearrangement in both osteoblasts and osteoclasts was altered, with a loss of integrity of the F-actin ring at the periphery of the osteoclasts. For the first time our studies demonstrate a direct role of FH in the maintenance of bone structure and function and is highlighted as a promising therapeutic target in bone diseases., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Age-related alteration of the immune system with aging, or immunosenescence, plays a major role in several age-associated conditions, including loss of bone integrity. Studies over the past several years have clearly established the immune system is chronically activated with advanced aging, termed inflammaging, and is characterized by elevated levels of proinflammatory cytokines in response to physiological or environmental cues that essentially result in an arrested immune system that maintains a low-level state of activation. This age-associated inflammation impacts several biological systems including the innate immune system, where aging results in a skewing of the hematopoiesis toward the myeloid lineage, including the expansion of myeloid-derived suppressor cells (MDSCs). This heterogeneous population of myeloid cells classically displays immunosuppressive capacity but they also have the ability to directly differentiate into osteoclasts. This review explores the possibility of inflammaging to be involved in reduction of bone microarchitecture and loss of bone mass/strength through the expansion of MDSCs and the osteoclastogenic capacity and activity.

Methamphetamine (METH) is a drug of abuse, the acute and chronic use of which induces neurotoxic responses in the human brain, ultimately leading to neurocognitive disorders. Our goals were to understand the impact of METH on microglial mitochondrial respiration and to determine whether METH induces the activation of the mitochondrial-dependent intrinsic apoptosis pathway in microglia. We assessed the expression of pro- apoptosis genes using qPCR of RNA extracted from a human microglial cell line (HTHU). We examined the apoptosis-inducing effects of METH on microglial cells using digital holographic microscopy (DHM) to quantify real-time apoptotic volume decrease (AVD) in microglia in a noninvasive manner. METH treatment significantly increased AVD, activated Caspase 3/7, increased the gene expression levels of the pro- apoptosis proteins, APAF-1 and BAX, and decreased mitochondrial DNA content. Using immunofluorescence analysis, we found that METH increased the expression of the mitochondrial proteins cytochrome c and MCL-1, supporting the activation of mitochondrion-dependent (intrinsic) apoptosis pathway. Cellular bio-energetic flux analysis by Agilent Seahorse XF Analyzer revealed that METH treatment increased both oxidative and glycolytic respiration after 3 h, which was sustained for at least 24 h. Several events, such as oxidative stress, neuro-inflammatory responses, and mitochondrial dysfunction, may converge to mediate METH-induced apoptosis of microglia that may contribute to neurotoxicity of the CNS. Our study has important implications for therapeutic strategies aimed at preserving mitochondrial function in METH abusing patients.

Vitamin D insufficiency (serum 25-OH vitamin D < 30 ng/ml) affects 70-80% of the general population, yet the long-term impacts on physical performance and the progression of sarcopenia are poorly understood. We therefore followed 6-month-old male C57BL/6J mice ( n =6) consuming either sufficient (STD, 1000 IU) or insufficient (LOW, 125 IU) vitamin D3/kg chow for 12 months (equivalent to 20-30 human years). LOW supplemented mice exhibited a rapid decline of serum 25-OH vitamin D levels by two weeks that remained between 11-15 ng/mL for all time points thereafter. After 12 months LOW mice displayed worse grip endurance (34.6 ± 14.1 versus 147.5 ± 50.6 seconds, p=0.001), uphill sprint speed (16.0 ± 1.0 versus 21.8 ± 2.4 meters/min, p=0.0007), and stride length (4.4 ± 0.3 versus 5.1 ± 0.3, p=0.002). LOW mice also showed less lean body mass after 8 months (57.5% ± 5.1% versus 64.5% ± 4.0%, p=0.023), but not after 12 months of supplementation, as well as greater protein expression of atrophy pathway gene atrogin‑1. Additionally, microRNA sequencing revealed differential expression of mIR‑26a in muscle tissue of LOW mice. These data suggest chronic vitamin D insufficiency may be an important factor contributing to functional decline and sarcopenia.

Sarcopenia and frailty are highly prevalent in older individuals, increasing the risk of disability and loss of independence. High intensity interval training (HIIT) may provide a robust intervention for both sarcopenia and frailty by achieving both strength and endurance benefits with lower time commitments than other exercise regimens. To better understand the impacts of HIIT during aging, we compared 24-month-old C57BL/6J sedentary mice with those that were administered 10-minute uphill treadmill HIIT sessions three times per week over 16 weeks. Baseline and end point assessments included body composition, physical performance, and frailty based on criteria from the Fried physical frailty scale. HIIT-trained mice demonstrated dramatic improvement in grip strength (HIIT 10.9% vs -3.9% in sedentary mice), treadmill endurance (32.6% vs -2.0%), and gait speed (107.0% vs 39.0%). Muscles from HIIT mice also exhibited greater mass, larger fiber size, and an increase in mitochondrial biomass. Furthermore, HIIT exercise led to a dramatic reduction in frailty scores in five of six mice that were frail or prefrail at baseline, with four ultimately becoming nonfrail. The uphill treadmill HIIT exercise sessions were well tolerated by aged mice and led to performance gains, improvement in underlying muscle physiology, and reduction in frailty.

Osteoporosis, a disease characterized by progressive bone loss and increased risk of fracture, often results from menopausal loss of estrogen in women. Neuropeptide Y has been shown to negatively regulate bone formation, with amygdala specific deletion of the Y2 receptor resulting in increased bone mass in mice. In this study, ovariectomized (OVX) mice were injected once daily with JNJ-31020028, a brain penetrant Y2 receptor small molecule antagonist to determine the effects on bone formation. Antagonist treated mice had reduced weight and showed increased whole-body bone mineral density compared to vehicle-injected mice. Micro computerized tomography (micro-CT) demonstrated increased vertebral trabecular bone volume, connectivity density and trabecular thickness. Femoral micro-CT analysis revealed increased bone volume within trabecular regions and greater trabecular number, without significant difference in other parameters or within cortical regions. A decrease was seen in serum P1NP, a measure used to confirm positive treatment outcomes in bisphosphonate treated patients. C-terminal telopeptide 1 (CTX-1), a blood biomarker of bone resorption, was decreased in treated animals. The higher bone mineral density observed following Y2 antagonist treatment, as determined by whole-body DEXA scanning, is indicative of either enhanced mineralization or reduced bone loss. Additionally, our findings that ex vivo treatment of bone marrow cells with the Y2 antagonist did not affect osteoblast and osteoclast formation suggests the inhibitor is not affecting these cells directly, and suggests a central role for compound action in this system. Our results support the involvement of Y2R signalling in bone metabolism and give credence to the hypothesis that selective pharmacological manipulation of Y2R may provide anabolic benefits for treating osteoporosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Vitamin D insufficiency (serum 25-OH vitamin D > 10 ng/ml and < 30 ng/ml) is prevalent in the obese (body mass index (BMI) > 30 kg/m 2 ), yet relationships between the two are poorly understood. Objectives of this study include identification of the impact of obesity on reducing serum 25-OH vitamin D concentration, particularly in response to altered vitamin D 3 supplementation, and to elucidate the longitudinal impact of serum 25-OH vitamin D on body mass index., Methods: Twenty four-week-old lean and obese male C57BL/6 J mice were fed low, standard, or high levels of cholecalciferol supplementation and followed for 24 weeks. Longitudinal measurements include serum 25-OH and 1,25-(OH) 2 vitamin D, intact PTH, and calcium concentrations, as well as BMI, bone density and body fat/lean mass., Results: Baseline serum 25-OH concentrations were not different in lean and obese mice (lean 32.8 ± 4.4 ng/ml versus obese 30.9 ± 1.6 ng/ml p = 0.09). Lean mice receiving low supplementation exhibited rapid declines in serum 25-OH vitamin D concentrations, falling from 33.4 ± 5.4 ng/ml to 14.5 ± 3.4 ng/ml after 2 weeks, while obese mice declined at a lower rate, falling from 30.9 ± 1.5 to 19.0 ± 0.9 ng/ml within the same time period. Surprisingly, high vitamin D 3 supplementation did not substantially increase serum vitamin D concentrations above standard supplementation, in either lean or obese mice. No differences in serum 1,25-(OH) 2 vitamin D, intact parathyroid hormone (PTH) or serum calcium were observed between lean and obese mice within the same vitamin D supplementation group. Yet obese mice exhibited lower serum calcitriol, higher serum PTH, and lower bone mineral density (BMD) than did lean mice. Additionally, neither body mass index nor body fat % was significantly correlated with vitamin D concentrations. Interestingly, lean mice with high vitamin D supplementation consumed significantly more food than did lean mice with standard or low supplementation (14.6 ± 1.7 kcal/mouse/day versus 11.8 ± 1.4 and 12.3 ± 1.7 respectively, p  < 0.0001 for both)., Conclusions: Low cholecalciferol supplementation in both lean and obese mice significantly and sustainably reduces serum 25-OH vitamin D concentrations. Interestingly, obesity slowed the rate of decline. Over the period of the study, vitamin D insufficiency was not subsequently correlated with greater BMI/body fat, although lean mice with high supplementation consumed greater calories with no apparent BMI increase.

Frailty is highly prevalent in the elderly, increasing the risk of poor outcomes that include falls, incident disability, hospitalization, and mortality. Thus, a great need exists to characterize the underlying mechanisms and ultimately identify strategies that prevent, delay, and even reverse frailty. Mouse models can provide insight into molecular mechanisms of frailty by reducing variability in lifestyle and genetic factors that can complicate interpretation of human clinical data. Frailty, generally recognized as a syndrome involving reduced homeostatic reserve in response to physiologic challenges and increasing susceptibility to poor health outcomes, is predominantly assessed using two independent strategies, integrated phenotype and deficit accumulation. The integrated phenotype defines frailty by the presentation of factors affecting functional capacity such as weight loss, exhaustion, low activity levels, slow gait, and grip strength. The deficit accumulation paradigm draws parameters from a greater range of physiological systems, such as the ability to perform daily activities, coordination and gait, mental components, physiological problems, and history and presence of medical morbidities. This strategic division also applies within the emerging field of mouse frailty models, with both methodologies showing usefulness in providing insight into physiologic mechanisms and testing interventions. Our review will explore the strategies used, caveats in methodology, and future directions in the application of animal models for the study of the frailty syndrome.

The BclXL apoptotic repressor bears the propensity to associate into megadalton oligomers in solution, particularly under acidic pH. Herein, using various biophysical methods, we analyze the effect of temperature on the oligomerization of BclXL. Our data show that BclXL undergoes irreversible aggregation and assembles into highly-ordered rope-like homogeneous fibrils with length in the order of mm and a diameter in the μm-range under elevated temperatures. Remarkably, the formation of such fibrils correlates with the decay of a largely α-helical fold into a predominantly β-sheet architecture of BclXL in a manner akin to the formation of amyloid fibrils. Further interrogation reveals that while BclXL fibrils formed under elevated temperatures show no observable affinity toward BH3 ligands, they appear to be optimally primed for insertion into cardiolipin bicelles. This salient observation strongly argues that BclXL fibrils likely represent an on-pathway intermediate for insertion into mitochondrial outer membrane during the onset of apoptosis. Collectively, our study sheds light on the propensity of BclXL to form amyloid-like fibrils with important consequences on its mechanism of action in gauging the apoptotic fate of cells in health and disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment., (© 2013 FEBS.)

Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Allostery plays a key role in dictating the stoichiometry and thermodynamics of multi-protein complexes driving a plethora of cellular processes central to health and disease. Herein, using various biophysical tools, we demonstrate that although Sos1 nucleotide exchange factor and Gab1 docking protein recognize two non-overlapping sites within the Grb2 adaptor, allostery promotes the formation of two distinct pools of Grb2-Sos1 and Grb2-Gab1 binary signaling complexes in concert in lieu of a composite Sos1-Grb2-Gab1 ternary complex. Of particular interest is the observation that the binding of Sos1 to the nSH3 domain within Grb2 sterically blocks the binding of Gab1 to the cSH3 domain and vice versa in a mutually exclusive manner. Importantly, the formation of both the Grb2-Sos1 and Grb2-Gab1 binary complexes is governed by a stoichiometry of 2:1, whereby the respective SH3 domains within Grb2 homodimer bind to Sos1 and Gab1 via multivalent interactions. Collectively, our study sheds new light on the role of allostery in mediating cellular signaling machinery., (Copyright © 2013 John Wiley & Sons, Ltd.)

Solution pH is believed to serve as an intricate regulatory switch in the induction of apoptosis central to embryonic development and cellular homeostasis. Herein, using an array of biophysical techniques, we provide evidence that acidic pH promotes the assembly of BclXL apoptotic repressor into a megadalton oligomer with a plume-like appearance and harboring structural features characteristic of a molten globule. Strikingly, our data reveal that pH tightly modulates not only oligomerization but also ligand binding and membrane insertion of BclXL in a highly subtle manner. Thus, while oligomerization and the accompanying molten globular content of BclXL is least favorable at pH 6, both of these structural features become more pronounced under acidic and alkaline conditions. However, membrane insertion of BclXL appears to be predominantly favored under acidic conditions. In a remarkable contrast, while ligand binding to BclXL optimally occurs at pH 6, it is diminished by an order of magnitude at lower and higher pH. This reciprocal relationship between BclXL oligomerization and ligand binding lends new insights into how pH modulates functional versatility of a key apoptotic regulator and strongly argues that the molten globule may serve as an intermediate primed for membrane insertion in response to apoptotic cues., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The WW-containing oxidoreductase (WWOX) tumor suppressor participates in a diverse array of cellular activities by virtue of its ability to recognize WW-binding protein 1 (WBP1) and WW-binding protein 2 (WBP2) signaling adaptors among a wide variety of other ligands. Herein, using a multitude of biophysical techniques, we provide evidence that while the WW1 domain of WWOX binds to PPXY motifs within WBP1 and WBP2 in a physiologically relevant manner, the WW2 domain exhibits no affinity toward any of these PPXY motifs. Importantly, our data suggest that while R25/W44 residues located within the binding pocket of a triple-stranded β-fold of WW1 domain are critical for the recognition of PPXY ligands, they are replaced by the chemically distinct E66/Y85 duo at structurally equivalent positions within the WW2 domain, thereby accounting for its failure to bind PPXY ligands. Predictably, not only does the introduction of E66R/Y85W double substitution within the WW2 domain result in gain of function but the resulting engineered domain, hereinafter referred to as WW2&#95;RW, also appears to be a much stronger binding partner of WBP1 and WBP2 than the wild-type WW1 domain. We also show that while the WW1 domain is structurally disordered and folds upon ligand binding, the WW2 domain not only adopts a fully structured conformation but also aids stabilization and ligand binding to WW1 domain. This salient observation implies that the WW2 domain likely serves as a chaperone to augment the physiological function of WW1 domain within WWOX. Collectively, our study lays the groundwork for understanding the molecular basis of a key protein-protein interaction pertinent to human health and disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although the growth factor receptor binder 2 (Grb2)-Grb2-associated binder (Gab)1 macromolecular complex mediates a multitude of cellular signaling cascades, the molecular basis of its assembly has hitherto remained largely elusive. Herein, using an array of biophysical techniques, we show that, whereas Grb2 exists in a monomer-dimer equilibrium, the proline-rich (PR) domain of Gab1 is a monomer in solution. Of particular interest is the observation that although the PR domain appears to be structurally disordered, it nonetheless adopts a more or less compact conformation reminiscent of natively folded globular proteins. Importantly, the structurally flexible conformation of the PR domain appears to facilitate the binding of Gab1 to Grb2 with a 1:2 stoichiometry. More specifically, the formation of the Grb2-Gab1 signaling complex is driven via a bivalent interaction through the binding of the C-terminal homology 3 (cSH3) domain within each monomer of Grb2 homodimer to two distinct RXXK motifs, herein designated G1 and G2, located within the PR domain of Gab1. Strikingly, in spite of the key role of bivalency in driving this macromolecular assembly, the cSH3 domains bind to the G1 and G2 motifs in an independent manner with zero cooperativity. Taken together, our findings shed new light on the physicochemical forces driving the assembly of a key macromolecular signaling complex that is relevant to cellular health and disease., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Despite its key role in driving cellular growth and proliferation through receptor tyrosine kinase (RTK) signaling, the Grb2-Sos1 macromolecular interaction remains poorly understood in mechanistic terms. Herein, using an array of biophysical methods, we provide evidence that although the Grb2 adaptor can potentially bind to all four PXψPXR motifs (designated herein S1-S4) located within the Sos1 guanine nucleotide exchange factor, the formation of the Grb2-Sos1 signaling complex occurs with a 2:1 stoichiometry. Strikingly, such bivalent binding appears to be driven by the association of the Grb2 homodimer to only two of four potential PXψPXR motifs within Sos1 at any one time. Of particular interest is the observation that of a possible six pairwise combinations in which S1-S4 motifs may act in concert for the docking of the Grb2 homodimer through bivalent binding, only S1 and S3, S1 and S4, S2 and S4, and S3 and S4 do so, while pairwise combinations of sites S1 and S2 and sites S2 and S3 appear to afford only monovalent binding. This salient observation implicates the role of local physical constraints in fine-tuning the conformational heterogeneity of the Grb2-Sos1 signaling complex. Importantly, the presence of multiple binding sites within Sos1 appears to provide a physical route for Grb2 to hop in a flip-flop manner from one site to the next through facilitated diffusion, and such rapid exchange forms the basis of positive cooperativity driving the bivalent binding of Grb2 to Sos1 with high affinity. Collectively, our study sheds new light on the assembly of a key macromolecular signaling complex central to cellular machinery in health and disease.

B-cell lymphoma extra large (BclXL) apoptotic repressor plays a central role in determining the fate of cells to live or die during physiological processes such as embryonic development and tissue homeostasis. Herein, using a myriad of biophysical techniques, we provide evidence that ligand binding and membrane insertion compete with oligomerization of BclXL in solution. Of particular importance is the observation that such oligomerization is driven by the intermolecular binding of its C-terminal transmembrane (TM) domain to the canonical hydrophobic groove in a domain-swapped trans fashion, whereby the TM domain of one monomer occupies the canonical hydrophobic groove within the other monomer and vice versa. Binding of BH3 ligands to the canonical hydrophobic groove displaces the TM domain in a competitive manner, allowing BclXL to dissociate into monomers upon hetero-association. Remarkably, spontaneous insertion of BclXL into DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine) bicelles results in a dramatic conformational change such that it can no longer recognize the BH3 ligands in what has come to be known as the "hit-and-run" mechanism. Collectively, our data suggest that oligomerization of a key apoptotic repressor serves as an allosteric switch that fine-tunes its ligand binding and membrane insertion pertinent to the regulation of apoptotic machinery., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery and circular dichroism in combination with molecular modeling and molecular dynamics, we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, nonconsensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a nonbulky and flexible glycine one residue to the C-terminal side of the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, suggesting that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

Estrogen receptor α (ERα) acts as a transcription factor by virtue of the ability of its DNA-binding (DB) domain, comprised of a tandem pair of zinc fingers, to recognize the estrogen response element within the promoters of target genes. Herein, using an array of biophysical methods, we probe the structural consequences of the replacement of zinc within the DB domain of ERα with various environmental metals and their effects on the thermodynamics of binding to DNA. Our data reveal that whereas the DB domain reconstituted with divalent ions of zinc, cadmium, mercury, and cobalt binds to DNA with affinities in the nanomolar range, divalent ions of barium, copper, iron, lead, manganese, nickel, and tin are unable to regenerate DB domain with DNA-binding potential, although they can compete with zinc for coordinating the cysteine ligands within the zinc fingers. We also show that the metal-free DB domain is a homodimer in solution and that the binding of various metals only results in subtle secondary and tertiary structural changes, implying that metal coordination may only be essential for binding to DNA. Collectively, our findings provide mechanistic insights into how environmental metals may modulate the physiological function of a key nuclear receptor involved in mediating a plethora of cellular functions central to human health and disease., (Copyright © 2011 John Wiley & Sons, Ltd.)

A ubiquitous component of cellular signaling machinery, Gab1 docker plays a pivotal role in routing extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. Here, using isothermal titration calorimetry (ITC) in combination with macromolecular modeling (MM), we show that although Gab1 contains four distinct RXXK motifs, designated G1, G2, G3, and G4, only G1 and G2 motifs bind to the cSH3 domain of Grb2 adaptor and do so with distinct mechanisms. Thus, while the G1 motif strictly requires the PPRPPKP consensus sequence for high-affinity binding to the cSH3 domain, the G2 motif displays preference for the PXVXRXLKPXR consensus. Such sequential differences in the binding of G1 and G2 motifs arise from their ability to adopt distinct polyproline type II (PPII)- and 3(10) -helical conformations upon binding to the cSH3 domain, respectively. Collectively, our study provides detailed biophysical insights into a key protein-protein interaction involved in a diverse array of signaling cascades central to health and disease., (Copyright © 2010 John Wiley & Sons, Ltd.)

Although allostery plays a central role in driving protein-DNA interactions, the physical basis of such cooperative behavior remains poorly understood. In the present study, using isothermal titration calorimetry in conjunction with site-directed mutagenesis, we provide evidence that an intricate network of energetically-coupled residues within the basic regions of the Jun-Fos heterodimeric transcription factor accounts for its allosteric binding to DNA. Remarkably, energetic coupling is prevalent in residues that are both close in space, as well as residues distant in space, implicating the role of both short- and long-range cooperative interactions in driving the assembly of this key protein-DNA interaction. Unexpectedly, many of the energetically-coupled residues involved in orchestrating such a cooperative network of interactions are poorly conserved across other members of the basic zipper family, emphasizing the importance of basic residues in dictating the specificity of basic zipper-DNA interactions. Collectively, our thermodynamic analysis maps an allosteric communication channel driving a key protein-DNA interaction central to cellular functions in health and disease., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Upon binding to estrogens, the ERα nuclear receptor acts as a transcription factor and mediates a multitude of cellular functions central to health and disease. Herein, using isothermal titration calorimetry (ITC) and circular dichroism (CD) in conjunction with molecular modeling (MM), we analyze the effect of symmetric introduction of single nucleotide variations within each half-site of the estrogen response element (ERE) on the binding of ERα nuclear receptor. Our data reveal that ERα exudes remarkable tolerance and binds to all genetic variants in the physiologically relevant nanomolar-micromolar range with the consensus ERE motif affording the highest affinity. We provide rationale for how genetic variations within the ERE motif may reduce its affinity for ERα by orders of magnitude at atomic level. Our data also suggest that the introduction of genetic variations within the ERE motif allows it to sample a much greater conformational space. Surprisingly, ERα displays no preference for binding to ERE variants with higher AT content, implying that any advantage due to DNA plasticity may be largely compensated by unfavorable entropic factors. Collectively, our study bears important consequences for how genetic variations within DNA promoter elements may fine-tune the physiological action of ERα and other nuclear receptors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bulky DNA damage is corrected by the nucleotide excision repair (NER) pathway. Although the core biochemical mechanism of NER is understood, details including lesion recognition and repair in the context of chromatin remain to be elucidated. As more data become available, the complexity of lesion recognition in chromatin is becoming clear. This review will discuss current knowledge of DNA damage recognition in the context of chromatin, with a focus on the roles of chromatin remodeling and the specific lesion recognition protein DDB2 (DNA damage-binding protein 2) in chromatin repair. Additionally, we propose a model that ubiquitination-mediated DDB2 dissociation from chromatin, not its degradation, is important for GG-NER progression.

Nuclear receptors act as ligand-modulated transcription factors and orchestrate a plethora of cellular functions central to health and disease. Although studied for more than half a century, many mysteries surrounding the mechanism of action of nuclear receptors remain unresolved. Herein, using isothermal titration calorimetry (ITC) in conjunction with macromolecular modeling (MM), we provide evidence that the binding of the ERalpha nuclear receptor to its DNA response element is coupled to proton uptake by two ionizable residues, H196 and E203, located at the protein-DNA interface. Alanine substitution of these ionizable residues decouples protonation and hampers the binding of ERalpha to DNA by nearly 1 order of magnitude. Remarkably, H196 and E203 are predominantly conserved across approximately 50 members of the nuclear receptor family, implying that proton-coupled equilibrium may serve as a key regulatory switch for modulating protein-DNA interactions central to nuclear receptor function and regulation. Taken together, our findings unearth an unexpected but critical step in the molecular action of nuclear receptors and suggest that they may act as sensors of intracellular pH.

Leucine zippers, structural motifs typically comprised of five successive heptads of amino acids with a signature leucine at every seventh position, play a central role in the dimerization of bZIP family of transcription factors and their subsequent binding to the DNA promoter regions of target genes. Herein, using analytical laser scattering (ALS) in combination with isothermal titration calorimetry (ITC), we study the effect of successive C-terminal truncation of leucine zippers on the dimerization and energetics of binding of bZIP domains of Jun transcription factor to its DNA response element. Our data show that all five heptads are critical for the dimerization of bZIP domains and that the successive C-terminal truncation of residues leading up to each signature leucine significantly compromises the binding of bZIP domains to DNA. Taken together, our study provides novel insights into the energetic contributions of leucine zippers to the binding of bZIP domains of Jun transcription factor to DNA., (2010 Elsevier Inc. All rights reserved.)

Allostery has evolved as a form of local communication between interacting protein partners allowing them to quickly sense changes in their immediate vicinity in response to external cues. Herein, using isothermal titration calorimetry (ITC) in conjunction with circular dichroism (CD) and macromolecular modeling (MM), we show that the binding of Grb2 adaptor--a key signaling molecule involved in the activation of Ras GTPase--to its downstream partners Sos1 guanine nucleotide exchange factor and Gab1 docker is under tight allosteric regulation. Specifically, our findings reveal that the binding of one molecule of Sos1 to the nSH3 domain allosterically induces a conformational change within Grb2 such that the loading of a second molecule of Sos1 onto the cSH3 domain is blocked and, in so doing, allows Gab1 access to the cSH3 domain in an exclusively non-competitive manner to generate the Sos1-Grb2-Gab1 ternary signaling complex., (2009 Elsevier Inc. All rights reserved.)

Limb-bud and heart (LBH) is a key transcriptional regulator in vertebrates with pivotal roles in embryonic development and human disease. Herein, using a diverse array of biophysical techniques, we report the first structural characterization of LBH pertinent to its biological function. Our data reveal that LBH is structurally disordered with no discernable secondary or tertiary structure and exudes rod-like properties in solution. Consistent with these observations, we also demonstrate that LBH is conformationally flexible and thus may be capable of adapting distinct conformations under specific physiological contexts. We propose that LBH is a member of the intrinsically disordered protein (IDP) family, and that conformational plasticity may play a significant role in modulating LBH-dependent transcriptional processes., (Copyright 2009 Elsevier Inc. All rights reserved.)

The Jun-Fos heterodimeric transcription factor is a target of a diverse array of signaling cascades that initiate at the cell surface and converge in the nucleus and ultimately result in the expression of genes involved in a multitude of cellular processes central to health and disease. Here, using isothermal titration calorimetry in conjunction with circular dichroism, we report the effect of introducing single nucleotide variations within the TGACGTCA canonical motif on the binding of bZIP domains of Jun-Fos heterodimer to DNA. Our data reveal that the Jun-Fos heterodimer exhibits differential energetics in binding to such genetic variants in the physiologically relevant micromolar to submicromolar range with the TGACGTCA canonical motif affording the highest affinity. Although binding energetics are largely favored by enthalpic forces and accompanied by entropic penalty, neither the favorable enthalpy nor the unfavorable entropy correlates with the overall free energy of binding in agreement with the enthalpy-entropy compensation phenomenon widely observed in biological systems. However, a number of variants including the TGACGTCA canonical motif bind to the Jun-Fos heterodimer with high affinity through having overcome such enthalpy-entropy compensation barrier, arguing strongly that better understanding of the underlying invisible forces driving macromolecular interactions may be the key to future drug design. Our data also suggest that the Jun-Fos heterodimer has a preference for binding to TGACGTCA variants with higher AT content, implying that the DNA plasticity may be an important determinant of protein-DNA interactions. This notion is further corroborated by the observation that the introduction of genetic variations within the TGACGTCA motif allows it to sample a much greater conformational space. Taken together, these new findings further our understanding of the role of DNA sequence and conformation on protein-DNA interactions in thermodynamic terms.

Ubiquitously encountered in a wide variety of cellular processes, the Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites containing PXpsiPXR motifs within Sos1. Here, using isothermal titration calorimetry, we demonstrate that while the nSH3 domain binds with affinities in the physiological range to all four sites containing PXpsiPXR motifs, designated S1, S2, S3, and S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3, and S4 sites, the PXpsiPXR motif at the S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In striking contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site, but their role is not critical for the recognition of S2, S3, and S4 sites. Site-directed mutagenesis suggests that the two additional arginine residues flanking the PXpsiPXR motif at the S1 site contribute to free energy of binding via the formation of salt bridges with specific acidic residues in SH3 domains. Molecular modeling is employed to project these novel findings into the 3D structures of SH3 domains in complex with a peptide containing the PXpsiPXR motif and flanking arginine residues at the S1 site. Taken together, this study furthers our understanding of the assembly of a key signaling complex central to cellular machinery.

The Jun-Fos heterodimeric transcription factor is the terminal link between the transfer of extracellular information in the form of growth factors and cytokines to the site of DNA transcription within the nucleus in a wide variety of cellular processes central to health and disease. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TGACTCA cis element and all possible single nucleotide variants thereof encountered widely within the promoters of a diverse array of genes. Our data show that Jun-Fos heterodimer tolerates single nucleotide substitutions and binds to TGACTCA variants with affinities in the physiologically relevant micromolar to submicromolar range. The energetics of binding are richly favored by enthalpic forces and opposed by entropic changes across the entire spectrum of TGACTCA variants in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific TGACTCA variants in a preferred orientation. Our 3D atomic models reveal that such orientational preference results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues R263 and K148 located within the basic regions of Jun and Fos, respectively. Taken together, our data suggest that the single nucleotide variants of the TGACTCA motif modulate energetics and orientation of binding of the Jun-Fos heterodimer and that such behavior may be a critical determinant of differential regulation of specific genes under the control of this transcription factor. Our study also bears important consequences for the occurrence of single nucleotide polymorphisms within the TGACTCA cis element at specific gene promoters between different individuals.

The Jun oncoprotein belongs to the AP1 family of transcription factors that is collectively engaged in diverse cellular processes by virtue of their ability to bind to the promoters of a wide spectrum of genes in a DNA sequence-dependent manner. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domain of Jun to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of Jun to both sites occurs with indistinguishable affinities but with distinct thermodynamic signatures comprised of favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures. Furthermore, anomalously large negative heat capacity changes observed provoke a model in which Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. Taken together, our data provide novel insights into the energetics of a key protein-DNA interaction pertinent to cellular signaling and cancer. Our study underscores the notion that the folding and dimerization of transcription factors upon association with DNA may be a more general mechanism employed in protein-DNA interactions and that the conventional school of thought may need to be re-evaluated.

Grb2-Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and C-terminal SH3 (cSH3) domains of Grb2 to a proline-rich sequence in Sos1, provides a key regulatory switch that relays signaling from activated receptor tyrosine kinases to downstream effector molecules such as Ras. Here, using isothermal titration calorimetry in combination with site-directed mutagenesis, we show that the nSH3 domain binds to a Sos1-derived peptide containing the proline-rich consensus motif PPVPPR with an affinity that is nearly threefold greater than that observed for the binding of cSH3 domain. We further demonstrate that such differential binding of nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue in the RT loop of the beta-barrel fold to engage in the formation of a salt bridge with the arginine residue in the consensus motif PPVPPR. While this role is fulfilled by an optimally positioned D15 in the nSH3 domain, the chemically distinct and structurally non-equivalent E171 substitutes in the case of the cSH3 domain. Additionally, our data suggest that salt tightly modulates the binding of both SH3 domains to Sos1 in a thermodynamically distinct manner. Our data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Our new findings are rationalized in the context of 3D structural models of SH3 domains in complex with the Sos1 peptide. Taken together, our study provides structural basis of the differential binding of SH3 domains of Grb2 to Sos1 and a detailed thermodynamic profile of this key protein-protein interaction pertinent to cellular signaling and cancer.

Jun and Fos are components of the AP1 family of transcription factors and bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. Here, using the powerful technique of isothermal titration calorimetry, we characterize the thermodynamics of heterodimerization of leucine zippers of Jun and Fos. Our data suggest that the heterodimerization of leucine zippers is driven by enthalpic forces with unfavorable entropy change at physiological temperatures. Furthermore, the basic regions appear to modulate the heterodimerization of leucine zippers and may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain alpha-helical conformations in isolation and that the formation of the native coiled-coil alpha-helical dimer is attained through a coupled folding-dimerization mechanism.

Grb2 is an adaptor protein that couples activated receptor tyrosine kinases to downstream effector molecules such as Ras and Akt. Despite being a central player in mitogenic signaling and a target for therapeutic intervention, the role of Grb2 oligomerization in cellular signaling is not well understood. Here, using the techniques of size-exclusion chromatography, mass spectrometry, analytical ultra-centrifugation and isothermal titration calorimetry, we demonstrate that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven without an unfavorable enthalpic change at physiological temperatures. Our data indicate that enthalpy and entropy of dimer dissociation are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the overall free energy. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change derived is much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon Grb2 dimerization, implying that Grb2 monomers undergo conformational rearrangement upon dimerization. 3D structural models of Grb2 dimer and monomers suggest strongly that such conformational rearrangement upon dimerization may arise from domain swapping. Taken together, our study provides novel insights into the role of Grb2 as an adaptor in cellular signaling circuitry and how Grb2 dimerization may impart high fidelity in signal transduction as well as lead to rapid signal amplification upon receptor stimulation.

In response to mitogenic stimuli, the heterodimeric transcription factor Jun-Fos binds to the promoters of a diverse array of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryogenic development and cancer. In so doing, Jun-Fos heterodimer regulates gene expression central to physiology and pathology of the cell in a specific and timely manner. Here, using the technique of isothermal titration calorimetry (ITC), we report detailed thermodynamics of the bZIP domains of Jun-Fos heterodimer to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of the bZIP domains to both TRE and CRE is under enthalpic control and accompanied by entropic penalty at physiological temperatures. Although the bZIP domains bind to both TRE and CRE with very similar affinities, the enthalpic contributions to the free energy of binding to CRE are more favorable than TRE, while the entropic penalty to the free energy of binding to TRE is smaller than CRE. Despite such differences in their thermodynamic signatures, enthalpy and entropy of binding of the bZIP domains to both TRE and CRE are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the free energy of binding. From the plot of enthalpy change versus temperature, the magnitude of heat capacity change determined is much larger than that expected from the direct association of bZIP domains with DNA. This observation is interpreted to suggest that the basic regions in the bZIP domains are largely unstructured in the absence of DNA and only become structured upon interaction with DNA in a coupled folding and binding manner. Our new findings are rationalized in the context of 3D structural models of bZIP domains of Jun-Fos heterodimer in complex with dsDNA oligos containing the TRE and CRE consensus sequences. Taken together, our study demonstrates that enthalpy is the major driving force for a key protein-DNA interaction pertinent to cellular signaling and that protein-DNA interactions with similar binding affinities may be accompanied by differential thermodynamic signatures. Our data corroborate the notion that the DNA-induced protein structural changes are a general feature of the bZIP family of transcription factors.