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Introduction: While 10% of pregnant individuals report a penicillin allergy, there is no established best practice for penicillin allergy delabeling in pregnancy. To better understand options for penicillin delabeling, we aimed to evaluate two penicillin allergy delabeling protocols in pregnancy regarding efficacy, adverse events, and patient satisfaction., Material and Methods: From July 2019 to December 2022, we completed a two-center prospective cohort study, where each site recruited pregnant patients over 24 weeks gestational age with a reported penicillin allergy. One center offered antepartum amoxicillin oral challenges, either directly or after negative skin testing (i.e., antepartum oral challenge site). Our other centers completed a two-step approach with antepartum penicillin skin testing only and deferred oral challenges to the postpartum period (i.e., postpartum oral challenge site). Our primary outcome was the rate of penicillin allergy delabeling, defined as tolerating an antibiotic challenge with penicillin or amoxicillin. Univariate analyses were completed using chi-squared, Fisher's exact, and Wilcoxon rank tests., Results: During the study period, 276 pregnant patients were assessed, with 207 in the antepartum oral challenge site and 69 in the postpartum oral challenge site. Among the 204 patients who completed antepartum oral challenges, 201 (98%) passed without reactions. Deferring oral challenges to the postpartum period led to a loss of follow-up for 37/53 (70%) of eligible individuals. Overall, 97% (201/207) of patients at the antepartum oral challenge site were delabeled from their penicillin allergy-compared to 38% (26/69) of patients referred to the postpartum oral challenge site (p < 0.0001). Three antepartum oral challenge reactions were noted, including two mild cutaneous reactions and a case of transient abdominal discomfort., Conclusions: Antepartum amoxicillin oral challenge is a more effective method to delabel pregnant patients from their penicillin allergy. Deferral of oral challenges to the postpartum period introduces a significant barrier for penicillin allergy delabeling., (© 2024 The Author(s). Acta Obstetricia et Gynecologica Scandinavica published by John Wiley & Sons Ltd on behalf of Nordic Federation of Societies of Obstetrics and Gynecology (NFOG).)

Temozolomide (TMZ) resistance in glioblastoma (GB) poses a significant therapeutic challenge. We developed a TMZ-resistant (TMZ-R) U251 GB model, revealing distinct differences in cell viability, apoptosis, autophagy, and lipid metabolism between TMZ-R and non-resistant (TMZ-NR) cells. TMZ-NR cells exhibited heightened sensitivity to TMZ-induced apoptosis, while TMZ-R cells-maintained viability. Autophagy flux was completely inhibited in TMZ-R cells, indicated by LC3βII and SQSTM1 accumulation. BCL2L13, which showed higher expression in TMZ-R cells, demonstrated increased interaction with Ceramide Synthase 6 (CerS6) and reduced interaction with Ceramide Synthase 2 (CerS2) in TMZ-NR cells. BCL2L13 knockdown (KD) disrupted autophagy flux, decreasing autophagosome accumulation in TMZ-R cells while increasing it in TMZ-NR cells. These changes contributed to altered ceramide profiles, where TMZ-R cells displayed elevated levels of Cer 16:0, 18:0, 20:0, 22:0, 24:0, and 24:1. Our findings highlight BCL2L13 and altered ceramide metabolism as potential therapeutic targets to overcome TMZ resistance in GB.

Introduction: Cardiac arrest remains one of the most common causes of death with the majority occurring outside of hospitals (out of hospital cardiac arrest). Despite advancements in resuscitation management, approximately 50% of comatose cardiac arrest patients (CCAP) will suffer a severe unsurvivable brain injury. To assess brain injury, a neurological examination is conducted, however, its reliability in predicting outcomes in the first days following cardiac arrest is limited. Non-contrast CT is the most employed scan to assess hypoxic changes, even though it is not sensitive to early hypoxic-ischaemic changes in the brain. CT perfusion (CTP) has shown high sensitivity and specificity in brain death patients, although its use in predicting poor neurological outcome in CCAP has not yet been explored. The purpose of this study is to validate CTP for predicting poor neurological outcome (modified Rankin scale, mRS≥4) at hospital discharge in CCAP., Methods and Analysis: The CT Perfusion for Assessment of poor Neurological outcome in Comatose Cardiac Arrest Patients study is a prospective cohort study funded by the Manitoba Medical Research Foundation. Newly admitted CCAP receiving standard Targeted Temperature Management are eligible. Patients undergo a CTP at the same time as the admission standard of care head CT. Admission CTP findings will be compared with the reference standard of an accepted bedside clinical assessment at the time of admission. Deferred consent will be used. The primary outcome is a binary outcome of good neurological status, defined as mRs<4 or poor neurological status (mRs≥4) at hospital discharge. A total of 90 patients will be enrolled., Ethics and Dissemination: This study has been approved by the University of Manitoba Health Research Ethics Board. The findings from our study will be disseminated through peer-reviewed journals and presentations at local rounds, national and international conferences. The public will be informed at the end of the study., Trial Registration Number: NCT04323020., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: Termed the "silent epidemic," traumatic brain injury (TBI) is one of the greatest global contributors not only to post-traumatic death but also to post-traumatic long-term disability. This systematic review and meta-analysis aims to specifically evaluate the prognostic value of features on initial imaging completed within 24 h of arrival in adult patients with TBI., Method: The authors followed the PRISMA 2020 checklist for systematic review and meta-analysis design and reporting. Comprehensive searches of the Medline and Embase databases were carried out. Two independent readers extracted the following demographic, clinical and imaging information using a predetermined data abstraction form. Statistics were performed using Revman 5.4.1 and R version 4.2.0. For pooled data in meta-analysis, forest plots for sensitivity and specificity were created to calculate the diagnostic odds ratio (DOR). Summary receiver operating characteristic (SROC) curves were generated using a bivariate model, and diagnostic accuracy was determined using pooled sensitivity and specificity as well as the area under the receiver operator characteristic curve (AUC)., Results: There were 10,733 patients over the 19 studies. Overall, most of the studies included had high levels of bias in multiple, particularly when it came to selection bias in patient sampling, bias in controlling for confounders, and reporting bias, such as in reporting missing data. Only subdural hematoma (SDH) and mortality in all TBI patients had both an AUC with 95% CI not crossing 0.5 and a DOR with 95% CI not crossing 1, at 0.593 (95% CI: 0.556-0.725) and 2.755 (95% CI: 1.474-5.148), respectively., Conclusion: In meta-analysis, only SDH with mortality in all TBI patients had a moderate but significant association. Given the small number of studies, additional research focused on initial imaging, particularly for imaging modalities other than NECT, is required in order to confirm the findings of our meta-analysis and to further evaluate the association of imaging findings and outcome.

Immunometabolism plays a crucial role in the activation and functional plasticity of immune cells, which in large determines a variety of health and disease states. Factors that integrate immunometabolism in immune cell signaling and functions are beginning to be identified. Previously, we have reported that two transgenic mouse models, Mito-Ob and mutant Mito-Ob (m-Mito-Ob), overexpressing a pleiotropic protein, prohibitin (PHB) or a mutant form of PHB (Tyr114Phe-PHB or m-PHB), respectively, developed distinct immunometabolic phenotypes. Specifically, the immune phenotype appears to be driven by the monocytic cell lineage. Based on immunophenotyping of their splenocytes, we focused our attention on macrophages and hypothesized that PHB may play a role in regulating the two functionally polarized states, M1 and M2. Here, we report that macrophage polarization to the M1 and M2 phenotypes did not alter PHB protein level, but overexpression of PHB in macrophages differentially affected cytokine production in the two polarized states. Furthermore, we found that mutation of the Tyr 114 phosphorylation site in PHB affects ERK and STAT6 signaling, arginase synthesis and activity, and mitochondrial respiration in macrophages indicating an important role of PHB in integrating cell signaling events with cell metabolism. In summary, we have discovered that PHB is a crucial regulator in the functional plasticity of macrophages. These initial studies expect to lay the foundation for future research into the relationship between cell signaling events pertaining to immunometabolism in immune cell functions, which are integral components of immune-related health and disease., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hemorrhagic transformation is caused by extravasation of blood products from vessels after acute ischemic stroke. It is an undesirable and potentially devastating complication, which occurs in 10%-40% of clinical cases. Hemorrhagic transformation is classified into four subtypes based on European cooperative acute stroke study II. Predicting hemorrhagic complications at presentation can be useful life saving/altering decisions for the patient. Also, understanding the mechanisms of hemorrhagic transformation can lead to new treatments and intervention measures. We highlighted various imaging techniques that have been used to predict hemorrhagic transformation. Specifically, we looked at the usefulness of perfusion and permeability imaging for hemorrhagic transformation. Use of imaging to predict hemorrhagic transformation could change patient management that may lead to the prevention of hemorrhagic transformation before it occurs. We concluded that the current evidence is not strong enough to rely on these imaging parameters for predicting hemorrhagic transformation and more studies are required.

Introduction: Severe traumatic brain injury (TBI) is a catastrophic neurological condition with significant economic burden. Early in-hospital mortality (<48 hours) with severe TBI is estimated at 50%. Several clinical examinations exist to determine brain death; however, most are difficult to elicit in the acute setting in patients with severe TBI. Having a definitive assessment tool would help predict early in-hospital mortality in this population. CT perfusion (CTP) has shown promise diagnosing early in-hospital mortality in patients with severe TBI and other populations. The purpose of this study is to validate admission CTP features of brain death relative to the clinical examination outcome for characterizing early in-hospital mortality in patients with severe TBI., Methods and Analysis: The Early Diagnosis of Mortality using Admission CT Perfusion in Severe Traumatic Brain Injury Patients study, is a prospective cohort study in patients with severe TBI funded by a grant from the Canadian Institute of Health Research. Adults aged 18 or older, with evidence of a severe TBI (Glasgow Coma Scale score ≤8 before initial resuscitation) and, on mechanical ventilation at the time of imaging are eligible. Patients will undergo CTP at the time of first imaging on their hospital admission. Admission CTP compares with the reference standard of an accepted bedside clinical assessment for brainstem function. Deferred consent will be used. The primary outcome is a binary outcome of mortality (dead) or survival (not dead) in the first 48 hours of admission. The planned sample size for achieving a sensitivity of 75% and a specificity of 95% with a CI of ±5% is 200 patients., Ethics and Dissemination: This study has been approved by the University of Manitoba Health Research Ethics Board. The findings from our study will be disseminated through peer-reviewed journals and presentations at local rounds, national and international conferences. The public will be informed through forums at the end of the study., Trial Registration Number: NCT04318665., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Lung cells are constantly exposed to various internal and external stressors that disrupt protein homeostasis. To cope with these stimuli, cells evoke a highly conserved adaptive mechanism called the unfolded protein response (UPR). UPR stressors can impose greater protein secretory demands on the endoplasmic reticulum (ER), resulting in the development, differentiation, and survival of these cell types to meet these increasing functional needs. Dysregulation of the UPR leads to the development of the disease. The UPR and ER stress are involved in several human conditions, such as chronic inflammation, neurodegeneration, metabolic syndrome, and cancer. Furthermore, potent and specific compounds that target the UPR pathway are under development as future therapies. The focus of this review is to thoroughly describe the effects of both internal and external stressors on the ER in asthma. Furthermore, we discuss how the UPR signaling pathway is activated in the lungs to overcome cellular damage. We also present an overview of the pathogenic mechanisms, with a brief focus on potential strategies for pharmacological interventions.

The resident immune cells (e.g., macrophages) present in major metabolic tissues, such as adipose and liver tissues, play crucial roles in respective tissue homeostasis through cross talk with metabolic tissues, and consequently in metabolic homeostasis at the systemic level, and their dysregulation contributes to metabolic dysregulation at large, as well as many associated diseases. Moreover, the cross talk between different resident immune cells and metabolic tissues is not limited to an intra-organ level but also includes interorgan cross talk, as they work in a coordinated manner throughout the body, such as in adipose tissue, skeletal muscle, and liver. Thus, it is important to determine the impact of altered immune functions on metabolic homeostasis and vice versa, to enhance our knowledge of immunometabolic biology. Glucose and insulin tolerance tests are simple methods that enable the measurement and analysis of the overall glucose homeostasis at the systemic level. Here we describe the process of performing metabolic tests for glucose homeostasis in mice, as mouse models are often used for defining the mechanistic underpinnings of physiology and pathophysiology related to immunometabolism, and in preclinical studies.

Virus infection induces different cellular responses in infected cells. These include cellular stress responses like autophagy and unfolded protein response (UPR). Both autophagy and UPR are connected to programed cell death I (apoptosis) in chronic stress conditions to regulate cellular homeostasis via Bcl2 family proteins, CHOP and Beclin-1. In this review article we first briefly discuss arboviruses, influenza virus, and HIV and then describe the concepts of apoptosis, autophagy, and UPR. Finally, we focus upon how apoptosis, autophagy, and UPR are involved in the regulation of cellular responses to arboviruses, influenza virus and HIV infections. Abbreviation: AIDS: Acquired Immunodeficiency Syndrome; ATF6: Activating Transcription Factor 6; ATG6: Autophagy-specific Gene 6; BAG3: BCL Associated Athanogene 3; Bak: BCL-2-Anatagonist/Killer1; Bax; BCL-2: Associated X protein; Bcl-2: B cell Lymphoma 2x; BiP: Chaperon immunoglobulin heavy chain binding Protein; CARD: Caspase Recruitment Domain; cART: combination Antiretroviral Therapy; CCR5: C-C Chemokine Receptor type 5; CD4: Cluster of Differentiation 4; CHOP: C/EBP homologous protein; CXCR4: C-X-C Chemokine Receptor Type 4; Cyto c: Cytochrome C; DCs: Dendritic Cells; EDEM1: ER-degradation enhancing-a-mannosidase-like protein 1; ENV: Envelope; ER: Endoplasmic Reticulum; FasR: Fas Receptor;G2: Gap 2; G2/M: Gap2/Mitosis; GFAP: Glial Fibrillary Acidic Protein; GP120: Glycoprotein120; GP41: Glycoprotein41; HAND: HIV Associated Neurodegenerative Disease; HEK: Human Embryonic Kidney; HeLa: Human Cervical Epithelial Carcinoma; HIV: Human Immunodeficiency Virus; IPS-1: IFN-β promoter stimulator 1; IRE-1: Inositol Requiring Enzyme 1; IRGM: Immunity Related GTPase Family M protein; LAMP2A: Lysosome Associated Membrane Protein 2A; LC3: Microtubule Associated Light Chain 3; MDA5: Melanoma Differentiation Associated gene 5; MEF: Mouse Embryonic Fibroblast; MMP: Mitochondrial Membrane Permeabilization; Nef: Negative Regulatory Factor; OASIS: Old Astrocyte Specifically Induced Substrate; PAMP: Pathogen-Associated Molecular Pattern; PERK: Pancreatic Endoplasmic Reticulum Kinase; PRR: Pattern Recognition Receptor; Puma: P53 Upregulated Modulator of Apoptosis; RIG-I: Retinoic acid-Inducible Gene-I; Tat: Transactivator Protein of HIV; TLR: Toll-like receptor; ULK1: Unc51 Like Autophagy Activating Kinase 1; UPR: Unfolded Protein Response; Vpr: Viral Protein Regulatory; XBP1: X-Box Binding Protein 1.

Cardiovascular disease leading to heart failure (HF) remains a leading cause of morbidity and mortality worldwide. Improved pharmacological and interventional coronary procedures have led to improved outcomes following acute myocardial infarction. This success has translated into an unforeseen increased incidence in HF. This review summarizes the signaling pathways implicated in the transition to HF following cardiac injury. In addition, we provide an update on cell death signaling and discuss recent advances in cardiac fibrosis as an independent event leading to HF. Finally, we discuss cell-based therapies and their possible use to avert the deteriorating nature of HF. © 2019 American Physiological Society. Compr Physiol 9:75-125, 2019., (Copyright © 2019 American Physiological Society. All rights reserved.)

Background: Recently, we have developed a novel transgenic mouse model by overexpressing prohibitin (PHB) in adipocytes, which developed obesity due to upregulation of mitochondrial biogenesis in adipocytes, hence named "Mito-Ob." Interestingly, only male Mito-Ob mice developed obesity-related impaired glucose homeostasis and insulin sensitivity, whereas female Mito-Ob mice did not. The observed sex differences in metabolic dysregulation suggest a potential involvement of sex steroids. Thus, the main aim of this study is to investigate the role of sex steroids on the overall phenotype of Mito-Ob mice through gonadectomy, as well as direct effect of sex steroids on adipocytes from Mito-Ob mice in vitro., Methods: Mito-Ob mice and wild-type CD-1 mice were gonadectomized at 12 weeks of age. Age- and sex-matched sham-operated mice were used as controls. Body weight, white adipose tissue, glucose tolerance, and insulin sensitivity were analyzed 3 months post-surgery. Differentiation of adipocytes isolated from female and male Mito-Ob mice were studied with and without sex steroids., Results: Gonadectomy significantly reduced body weight in Mito-Ob mice compared with sham-operated mice, whereas the opposite trend was observed in wild-type mice. These changes occurred independent of food intake. A corresponding decrease in adipose tissue weight was found in gonadectomized Mito-Ob mice, but depot-specific differences were observed in male and female. Gonadectomy improved glucose tolerance in male wild-type and Mito-Ob mice, but the effect was more pronounced in wild-type mice. Gonadectomy did not alter insulin sensitivity in male Mito-Ob mice, but it was improved in male wild-type mice. In primary cell cultures, testosterone inhibited adipocyte differentiation to a lesser extent in male Mito-Ob preadipocytes compared with the wild-type mice. On the other hand, preadipocytes from female wild-type mice showed better differentiation potential than those from female Mito-Ob mice in the presence of 17β-estradiol., Conclusions: PHB requires sex steroids for the development of obese phenotype in Mito-Ob mice, which differentially affect glucose homeostasis and insulin sensitivity in male and female. It appears that PHB plays sex- and adipose depot-specific roles and involves additional factors. In vitro studies suggested that PHB differently influenced adipocyte differentiation in the presence and absence of sex steroids. Overall, this study along with available information in the literature indicated that a multifaceted relationship exists between PHB and sex steroids, which may work in a cell/tissue type- and sex-specific manner.

Despite advances in neurosurgical techniques and radio-/chemotherapy, the treatment of brain tumors remains a challenge. This is particularly true for the most frequent and fatal adult brain tumor, glioblastoma (GB). Upon diagnosis, the average survival time of GB patients remains only approximately 15months. The alkylating drug temozolomide (TMZ) is routinely used in brain tumor patients and induces apoptosis, autophagy and unfolded protein response (UPR). Here, we review these cellular mechanisms and their contributions to TMZ chemoresistance in brain tumors, with a particular emphasis on TMZ chemoresistance in glioma stem cells and GB., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Immunometabolism, which has important implications in cancer biology, has emerged as a major regulator of different immune cell types. Various factors that integrate metabolic switches within immune cells with signal directed program that promote or inhibit their functions remain largely unidentified. Furthermore, sex differences are known to exist in immune functions and cancer incidences in the body and sex steroid hormones are integral component of these differences. However, factors that mediate such differences, and the potential link between the two fundamental aspects of immune cell biology that contributes to sex differences in health and disease remain unexplored. New evidence derived from novel tissue-specific transgenic mouse models of prohibitin (PHB) has revealed its crucial role in sex differences in adipocyte and macrophage functions and a potential role in endocrine-immune crosstalk. This review provides a point of view on the emerging role of PHB in immune functions with special focus on immunometabolism and on the immunomodulatory effects of sex steroids. We propose that PHB plays a crucial role in integrating cell signaling events with metabolic switches, and may serve as a potential target for cancer immunotherapeutic., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Prohibitin is a pleiotropic protein that has roles in fundamental cellular processes, such as cellular proliferation and mitochondrial housekeeping, and in cell- or tissue-specific functions, such as adipogenesis and immune cell functions. The different functions of prohibitin are mediated by its cell compartment-specific attributes, which include acting as an adaptor molecule in membrane signaling, a scaffolding protein in mitochondria, and a transcriptional co-regulator in the nucleus. However, the precise relationship between its distinct cellular localization and diverse functions remain largely unknown. Accumulating evidence suggests that the phosphorylation of prohibitin plays a role in a number of cell signaling pathways and in intracellular trafficking. Herein, we discuss the known and potential importance of the site-specific phosphorylation of prohibitin in regulating these features. We will discuss this in the context of new evidence from tissue-specific transgenic mouse models of prohibitin, including a mutant prohibitin lacking a crucial tyrosine phosphorylation site. We conclude with the opinion that prohibitin can be used as a potential target for tyrosine kinase signal transduction-targeting therapy, including in insulin, growth factors, and immune signaling pathways., Competing Interests: The authors declare no conflict of interest.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and the most common cause of female infertility. However, its etiology and underlying mechanisms remain unclear. Here we report that a transgenic obese mouse (Mito-Ob) developed by overexpressing prohibitin in adipocytes develops polycystic ovaries. Initially, the female Mito-Ob mice were equally fertile to their wild-type littermates. The Mito-Ob mice began to gain weight after puberty, became significantly obese between 3-6 months of age, and ∼25% of them had become infertile by 9 months of age. Despite obesity, female Mito-Ob mice maintained glucose homeostasis and insulin sensitivity similar to their wild-type littermates. Mito-Ob mice showed morphologically distinct polycystic ovaries and elevated estradiol, but normal testosterone and insulin levels. Histological analysis of the ovaries showed signs of impaired follicular dynamics, such as preantral follicular arrest and reduced number, or absence, of corpus luteum. The ovaries of the infertile Mito-Ob mice were closely surrounded by periovarian adipose tissue, suggesting a potential role in anovulation. Collectively, these data suggest that elevated estradiol and obesity per se might lead to anovulation and polycystic ovaries independent of hyperinsulinemia and hyperandrogenism. As obesity often coexists with other abnormalities known to be involved in the development of PCOS such as insulin resistance, compensatory hyperinsulinemia and hyperandrogenism, the precise role of these factors in PCOS remains unclear. Mito-Ob mice provide an opportunity to study the effects of obesity on anovulation and ovarian cyst formation independent of the major drivers of obesity-linked PCOS., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The cholesterol biosynthesis pathway, also referred to as the mevalonate (MVA) pathway, is responsible for the biosynthesis of two key isoprenoids: farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Post-translational modification of small GTPases by FPP and GGPP has captured much attention due to their potential contribution to cancer, cardiovascular and neurodegenerative diseases. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) catalyzes the conversion of HMG-CoA to MVA, and is the rate-limiting step in the biosynthesis of cholesterol. Statins are HMGCR inhibitors that are used extensively in the treatment of hypercholesterolemia. Inhibitors of the MVA pathway exhibit anti-tumor effects and may reduce cancer incidence and cancer-related mortality in humans. In this review, we will focus on the mevalonate cascade and its regulation in cholesterol metabolism as well as polymorphisms of the MVA cascade in cancer development, infectious and cardiovascular disease (CVD)., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

A critical unmet need for the study of obesity-linked cancer is the lack of preclinical models that spontaneously develop obesity and cancer sequentially. Prohibitin (PHB) is a pleiotropic protein that has a role in adipose and immune functions. We capitalized on this attribute of PHB to develop a mouse model for obesity-linked tumor. We achieved this by expressing Y114F-PHB (m-PHB) from the aP2 gene promoter for simultaneous manipulation of adipogenic and immune signaling functions. The m-PHB mice develop obesity in a sex-neutral manner, but only male mice develop impaired glucose homeostasis and hyperinsulinemia similar to transgenic mice expressing PHB. Interestingly, only male m-PHB mice develop histiocytosis with lymphadenopathy, suggesting that metabolic dysregulation or m-PHB alone is not sufficient for the tumor development and that both are required for tumorigenesis. Moreover, ovariectomy in female m-PHB mice resulted in impaired glucose homeostasis, hyperinsulinemia and consequently tumor development similar to male m-PHB mice. These changes were not observed in sham-operated control m-Mito-Ob mice, further confirming the role of obesity-related metabolic dysregulation in tumor development in m-PHB mice. Our data provide a proof-of-concept that obesity-associated hyperinsulinemia promotes tumor development by facilitating dormant mutant to manifest and reveals a sex-dimorphic role of PHB in adipose-immune interaction or immunometabolism. Targeting PHB may provide a unique opportunity for the modulation of immunometabolism in obesity, cancer and in immune diseases.

Prohibitin (PHB) was discovered in a quest to find genes with antiproliferative functions. However, the attribute of PHB that is responsible for its antiproliferative function remains elusive. Meanwhile, recent studies have established PHB as a pleiotropic protein with roles in metabolism, immunity, and senescence. PHB has cell compartment-specific functions, acting as a scaffolding protein in mitochondria, an adaptor molecule in membrane signaling, and a transcriptional coregulator in the nucleus. However, it remains unclear whether different functions and locations of PHB are interrelated or independent from each other, or if PHB works in a tissue-specific manner. Here, we discuss new findings on the role of PHB in adipose-immune interaction and an unexpected role in sex differences in adipose and immune functions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sex differences are known to exist in adipose and immune functions in the body, and sex steroid hormones are known to be involved in sexually dimorphic biological and pathological processes related to adipose-immune interaction. However, our knowledge of proteins that mediate such differences is poor. Two novel obese mice models, Mito-Ob and m-Mito-Ob, that have been reported recently have revealed an unexpected role of a pleiotropic protein, prohibitin (PHB), in sex differences in adipose and immune functions. This discovery points towards a role of pleiotropic proteins and their potential interplay with sex steroid hormones in mediating sexually dimorphic adipose-immune interaction.

Obesity increases the risk for nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. However, the underlying mechanisms involved in the disease process remain unclear. Recently, we have developed a transgenic obese mouse model (Mito-Ob) by prohibitin mediated mitochondrial remodeling in adipocytes. The Mito-Ob mice develop obesity in a sex-neutral manner, but obesity-associated adipose inflammation and metabolic dysregulation in a male sex-specific manner. Here we report that with aging, the male Mito-Ob mice spontaneously develop obesity-linked NASH and hepatocellular carcinoma (HCC). In contrast, the female Mito-Ob mice maintained normal glucose and insulin levels and did not develop NASH and HCC. The anti-inflammatory peptide ghrelin was significantly upregulated in the female mice and down regulated in the male mice compared with respective control mice. In addition, a reduction in the markers of mitochondrial content and function was found in the liver of male Mito-Ob mice with NASH/HCC development. We found that ERK1/2 signaling was significantly upregulated whereas STAT3 signaling was significantly down regulated in the tumors from Mito-Ob mice. These data provide a proof-of-concept that the metabolic and inflammatory status of the adipose tissue and their interplay at the systemic and hepatic level play a central role in the pathogenesis of obesity-linked NASH and HCC.

The incidence of adult-onset T1D in low-risk non-HLA type has increased several folds, whereas the contemporaneous incidence in high-risk HLA-type remains stable. Various factors behind this selective increase in T1D in young adults remain unclear. Obesity and its associated abnormalities appear to be an important determinant; however, the underlying mechanism involved is not understood. Recently, we have developed two novel transgenic obese mice models, Mito-Ob and m-Mito-Ob, by expressing a pleiotropic protein prohibitin (PHB) and a phospho mutant form of PHB (Y114F-PHB or m-PHB) from the aP2 gene promoter, respectively. Both mice models develop obesity in a sex-neutral manner, independent of diet; but obesity associated chronic low-grade inflammation and insulin resistance in a male sex-specific manner. Interestingly, on a high fat diet (HFD) only male m-Mito-Ob mice displayed marked mononuclear cell infiltration in pancreas and developed insulitis that mimic adult-onset T1D. Male Mito-Ob mice that share the metabolic phenotype of male m-Mito-Ob mice, and female m-Mito-Ob that harbor m-PHB similar to male m-Mito-Ob mice, did not develop insulitis. Thus, insulitis development in male m-Mito-Ob in response to HFD requires both, obesity-related abnormalities and m-PHB. Collectively, this data provides a proof-of-concept that obesity-associated abnormalities couple environmental triggers with genetic susceptibility in adult-onset T1D and reveals PHB as a potential susceptibility gene for T1D., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Arboviruses are pathogens that widely affect the health of people in different communities around the world. Recently, a few successful approaches toward production of effective vaccines against some of these pathogens have been developed, but treatment and prevention of the resulting diseases remain a major health and research concern. The arbovirus infection and replication processes are complex, and many factors are involved in their regulation. Apoptosis, autophagy and the unfolded protein response (UPR) are three mechanisms that are involved in pathogenesis of many viruses. In this review, we focus on the importance of these pathways in the arbovirus replication and infection processes. We provide a brief introduction on how apoptosis, autophagy and the UPR are initiated and regulated, and then discuss the involvement of these pathways in regulation of arbovirus pathogenesis.

Mitochondria play vital roles in the maintenance of cellular homeostasis. They are a storehouse of cellular energy and antioxidative enzymes. Because of its immense role and function in the development of an organism, this organelle is required for the survival. Defects in mitochondrial proteins lead to complex mitochondrial disorders and heterogeneous diseases such as cancer, type 2 diabetes, and cardiovascular and neurodegenerative diseases. It is widely known in the literature that some of the mitochondrial proteins are regulated by posttranslational modifications. Hence, designing methods to assess these modifications in mitochondria will be an important way to study the regulatory roles of mitochondrial proteins in greater detail. In this chapter, we outlined procedures to isolate mitochondria from cells and separate the mitochondrial proteins by two-dimensional gel electrophoresis and identify the different posttranslational modifications in them by using antibodies specific to each posttranslational modification.

Adipocytes are the primary cells in the body that store excess energy as triglycerides. To perform this specialized function, adipocytes rely on their mitochondria; however, the role of adipocyte mitochondria in the regulation of adipose tissue homeostasis and its impact on metabolic regulation is not understood. We developed a transgenic mouse model, Mito-Ob, overexpressing prohibitin (PHB) in adipocytes. Mito-Ob mice developed obesity due to upregulation of mitochondrial biogenesis in adipocytes. Of note, Mito-Ob female mice developed more visceral fat than male mice. However, female mice exhibited no change in glucose homeostasis and had normal insulin and high adiponectin levels, whereas male mice had impaired glucose homeostasis, compromised brown adipose tissue structure, and high insulin and low adiponectin levels. Mechanistically, we found that PHB overexpression enhances the cross talk between the mitochondria and the nucleus and facilitates mitochondrial biogenesis. The data suggest a critical role of PHB and adipocyte mitochondria in adipose tissue homeostasis and reveal sex differences in the effect of PHB-induced adipocyte mitochondrial remodeling on whole-body metabolism. Targeting adipocyte mitochondria may provide new therapeutic opportunities for the treatment of obesity, a major risk factor for type 2 diabetes., (© 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs ('statins') directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD)), and cancer., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimer's diseases, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The posttranslational modification (PTM) in protein occurs in a regiospecific manner. In addition, the most commonly occurring PTMs involve similar residues in proteins such as acetylation, ubiquitylation, methylation and sumoylation at the lysine residue and phosphorylation and O-GlcNAc modification at serine/threonine residues. Thus, the possibility of modification sites where two such PTMs may occur in a mutually exclusive manner (ME-PTM) is much higher than known. A recent surge in the identification and the mapping of the commonly occurring PTMs in proteins has revealed that this is indeed the case. However, in what way such ME-PTM sites are regulated and what could be their relevance in the coordinated network of protein function remains to be known. To gain such potential insights in a biological context, we analyzed two most prevalent PTMs on the lysine residue by acetylation and ubiquitylation along with the most abundant PTM in proteins by phosphorylation among enzymes involved in glucose metabolism, a fundamental process in biology. The analysis of the PTM data sets has revealed two important clues that may be intrinsically associated with their regulation and function. First, the most commonly occurring PTMs by phosphorylation, acetylation and ubiquitylation are widespread and clustered in most of the enzymes involved in glucose metabolism; and the prevalence of phosphorylation sites correlates with the number of acetylation and ubiquitylation sites including the ME-modification sites. Second, the prevalence of ME-acetylation/ubiquitylation sites is exceptionally high among enzymes involved in glucose metabolism and have distinct pattern among the subset of enzymes of glucose metabolism such as glycolysis, tricarboxylic acid (TCA) cycle, glycogen synthesis, and the irreversible steps of gluconeogenesis. We hypothesize that phosphorylation including tyrosine phosphorylation plays an important role in the regulation of ME-acetylation/ubiquitylation sites and their similar pattern among the subset of functionally related proteins allows their coordinated regulation in the normal physiology. Similarly their coordinated dysregulation may underlie the disease processes such as reprogrammed metabolism in cancer, obesity, type 2 diabetes, and cardiovascular diseases. Our hypothesis provides an opportunity to understand the regulation of ME-PTMs in proteins and their relevance at the network level and is open for experimental validation.

Asthma is one of the fastest growing syndromes in many countries and is adding a huge cost to the health care system. Increasing reports have linked airway infectious diseases to asthma. Influenza is one of the most serious airway infectious diseases and in recent years there have been some serious influenza virus pandemics which caused increased fatality in numerous different populations. Diverse host response pathways during virus infection have been identified, including different cell death and survival pathways. These pathways include 1) programmed cell death I (apoptosis), 2) programmed cell death II (autophagy), and 3) endoplasmic reticulum stress with subsequent unfolded protein response (UPR). There has been extensive research on the regulatory roles of these pathways during the influenza virus life cycle. These studies address the benefits of enhancing or inhibiting these pathways on viral replication. Here we review the most recent and significant knowledge in this area for possible benefits to clinicians and basic scientist researchers in different areas of the respiratory and virology sciences.

The post-translational modification of protein by acetylation has been emerging as a prevalent modification in enzymes that catalyze intermediary metabolism. However, the dynamics of protein acetylation during adipocyte differentiation that involves a major shift in cellular metabolism is not known. In this study, we investigated the temporal changes in acetylation during adipocyte differentiation. Almost all acetylated proteins identified showed a sequential change in acetylation during the differentiation process. While the majority of the acetylated proteins showed a sequential upregulation during adipocyte differentiation, in a few proteins a sequential downregulation of protein acetylation was also observed. Our findings suggest that a wide-ranging temporal change in protein acetylation occurs during adipocyte differentiation including differentially expressed proteins signifying an important role in adipocyte differentiation.

Objective: To investigate whether prohibitin (PHB) is a target gene in adipocyte differentiation and modulates insulin-induced adipocyte differentiation., Design: 3T3-L1 preadipocyte overexpressing wild-type PHB and PHB mutant (lacking tyrosine-114 phosphorylation site) with or without insulin., Results: The treatment of 3T3-L1 fibroblasts with insulin or peroxisome proliferator-activated receptor-γ agonist resulted in an upregulation of PHB in a dose- and time-dependent manner. An analysis of the PHB promoter sequence revealed the presence of putative insulin-response elements and CCAAT/enhancer-binding protein transcription elements within ∼1 kb upstream of the translation initiation site. The functional relevance of these sites was determined using reporter gene assay. Surprisingly, PHB was also found to be regulated by leptin. Furthermore, the overexpression of PHB in 3T3-L1 fibroblasts was sufficient to induce adipogenesis., Conclusion: In summary, we have identified PHB as an important protein in adipocyte differentiation.

Background: Transglutaminase 2 (TG2) and its phosphorylation have been consistently found to be upregulated in a number of cancer cell types. At the molecular level, TG2 has been associated with the activation of nuclear factor-kappa B (NF-κB), protein kinase B (PKB/Akt) and in the downregulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, the underlying mechanism involved is not known. We have reported that protein kinase A (PKA) induced phosphorylation of TG2 at serine-216 (Ser(216)) regulates TG2 function and facilitates protein-protein interaction. However, the role of TG2 phosphorylation in the modulation of NF-κB, Akt and PTEN is not explored., Methods: In this study we have investigated the effect of TG2 phosphorylation on NF-κB, Akt and PTEN using embryonic fibroblasts derived from TG2 null mice (MEF(tg2-/-)) overexpressing native TG2 or mutant-TG2 (m-TG2) lacking Ser(216) phosphorylation site with and without dibutyryl cyclic-AMP (db-cAMP) stimulation. Functional consequences on cell cycle and cell motility were determined by fluorescence activated cell sorting (FACS) analysis and cell migration assay respectively., Results: PKA activation in TG2 overexpressing MEF(tg2-/-) cells resulted in an increased activation of NF-κB and Akt phosphorylation in comparison to empty vector transfected control cells as determined by the reporter-gene assay and immunoblot analysis respectively. These effects were not observed in MEF(tg2-/-) cells overexpressing m-TG2. Similarly, a significant downregulation of PTEN at both, the mRNA and protein levels were found in cells overexpressing TG2 in comparison to empty vector control and m-TG2 transfected cells. Furthermore, Akt activation correlated with the simultaneous activation of NF-κB and a decrease in PTEN suggesting that the facilitatory effect of TG2 on Akt activation occurs in a PTEN-dependent manner. Similar results were found with MCF-7 and T-47D breast cancer cells overexpressing TG2 and m-TG2 further supporting the role of TG2 phosphorylation in NF-κB activation and in the downregulation of PTEN., Conclusions: Collectively, these data suggest that phosphorylation of TG2 at Ser(216) plays a role in TG2 mediated activation of NF-κB, Akt and in the downregulation of PTEN. Blocking TG2 phosphorylation may provide a novel strategy to attenuate NF-κB activation and downregulation of PTEN in TG2 overexpressing cancers.

Transglutaminase 2 (TG2) is an enzyme with diverse biological functions. TG2 catalyzes transamidation reactions, has intrinsic kinase activity, and acts as a G-protein in intracellular signaling. TG2 (Tgm2)-null mice are glucose intolerant and have impaired glucose-stimulated insulin secretion (GSIS). Furthermore, three naturally occurring missense mutations in the human TGM2 gene, corresponding to amino acid substitutions of Met330Arg, Ile331Asn, and Asn333Ser in the TG2 protein, have been reported and found to be associated with early-onset type 2 diabetes. However, their effect on TG2 function is not fully understood. To determine this, we have reproduced naturally occurring mutations in TG2 using site-directed mutagenesis. Overexpression of Myc-TG2 mutants in INS-1E cells resulted in a reduction of GSIS in comparison with cells overexpressing wild-type Myc-TG2 (WT-TG2). The maximum reduction was found in cells overexpressing Ile331Asn-TG2 (32%) followed by Met330Arg-TG2 (20%), and the least in Asn333Ser-TG2 (7%). Enzymatic analysis revealed that TG2 mutants have impaired transamidation and kinase activities in comparison with WT-TG2. GTP-binding assays showed that TG2 mutants also have altered GTP-binding ability, which is found to be modulated in response to glucose stimulation. Collectively, these data suggest that naturally occurring mutations in TG2 affect transamidation, kinase, and GTP-binding functions of TG2. While reduced insulin secretion, as a result of naturally occurring mutations in TG2, is due to the impairment of more than one biological function of TG2, it is the transamidation function that appears to be impaired during the first phase, whereas the GTP-binding function affects the second phase of insulin secretion.

Simultaneous upregulation of transglutaminase 2 (TG2) and epidermal growth factor receptor (EGFR) have been reported in a number of systems. Moreover, TG2 has been identified as a downstream target gene for EGF/EGFR. However, it is not known whether the relationship between EGFR and TG2 is only one-way or collaborative. Using embryonic fibroblasts derived from TG2 null mice (MEF(tg2-/-)), co-overexpressing native TG2 and EGFR, here we report that TG2 differentially regulates EGFR protein in the presence and absence of EGF. In the absence of EGF, TG2 facilitates EGFR downregulation whereas in the presence of EGF, TG2 has opposite effect on EGFR and facilitates Akt phosphorylation. TG2 mediated ligand-independent downregulation of EGFR was not observed in MEF(tg2-/-) cells overexpressing Ser212Ala phospho mutant form of TG2 suggesting a role of TG2 phosphorylation in this process. However, similar to native TG2, Ser212Ala-TG2 mutant was also able to attenuate ligand-dependent down regulation of EGFR in MEF(tg2-/-) cells. Interestingly, overexpression of Ser216Ala-TG2 mutant led to downregulation of EGFR in MEF(tg2-/-) cells irrespective of the ligand. These results were further confirmed in breast cancer cells expressing high levels of EGFR. Collectively, data presented here suggests that the relationship between EGFR and TG2 is collaborative and phosphorylation of TG2 play a key role in it. Phospho mutant forms of TG2 reported in this study may be utilized as a part of a novel strategy to downregulate EGFR in cancers with enhanced EGFR signaling., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Autophagy is a very tightly regulated process that is important in many cellular processes including development, differentiation, survival and homoeostasis. The importance of this process has already been proven in numerous common diseases such as cancer and neurodegenerative disorders. Emerging data indicate that autophagy plays an important role in some liver diseases including liver injury induced by ischaemia reperfusion and alpha-1 antitrypsin Z allele-dependent liver disease. Autophagy may also occur in viral infection, and it may play a crucial role in antimicrobial host defence against pathogens, while supporting cellular homoeostasis processes. Here, the latest findings on the role of autophagy in viral hepatitis B and C infection, which are both serious health threats, will be reviewed., (© 2011 Blackwell Publishing Ltd.)

Post-translational modification of proteins at serine and threonine side chains by β-N-acetylglucosamine (O-GlcNAc) mediated by the enzyme β-N-acetylglucosamine transferase has been emerging as a fundamental regulatory mechanism encompassing a wide range of proteins involved in cell division, metabolism, transcription and cell signaling. Furthermore, an extensive interplay between O-GlcNAc modification and serine/threonine phosphorylation in a variety of proteins has been reported to exist. However, our understanding of the regulatory mechanisms involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins is still elusive. Recent success in the mapping of O-GlcNAc modification sites in proteins as a result of technological advancement in mass spectrometry have revealed two important clues which may be inherently connected to the regulation of O-GlcNAc modification and its interplay with phosphorylation in proteins. First, almost all O-GlcNAc modified proteins are known phospho proteins. Second, the prevalence of tyrosine phosphorylation among O-GlcNAc modified proteins is exceptionally higher (~68%) than its normal occurrence (~2%) alone. We hypothesize that phosphorylation may be a requisite for O-GlcNAc modification and tyrosine phosphorylation plays a role in the interplay between O-GlcNAc modification and serine/threonine phosphorylation in proteins. In other words, the interplay between O-GlcNAc modification and phosphorylation is not limited to serine/threonine phosphorylation but also includes tyrosine phosphorylation. Our hypothesis provides an opportunity to understand the underlying mechanism involved in O-GlcNAc modification and its interplay with serine/threonine phosphorylation in proteins. Furthermore, implication of our hypothesis extends to tyrosine kinase signaling.

Mitochondria are central to iron homeostasis. However, various proteins involved in iron metabolism inside the mitochondria are still to be identified. Herein we report that nuclear coded mitochondrial protein prohibitin binds to iron and involved in intracellular iron homeostasis. Like other iron regulated proteins, prohibitin mRNA contains functional iron-response element and is regulated by intracellular iron levels. Tyrosine residues involved in iron binding attribute of prohibitin are identified using site-directed mutagenesis. These data together suggest that prohibitin functions as an intracellular iron binding protein and plays a role in intracellular iron homeostasis., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Hyperglycemia induced increased posttranslational modification of proteins by O-linked-β-N-acetyl glucosamine (O-GlcNAcylation) and mitochondrial dysfunction has been independently implicated in the development of insulin resistance. It is not known whether repertoire of O-GlcNAcylated proteins includes mitochondrial proteins and their altered O-GlcNAcylation impinges on their phosphorylation mediated normal functioning thus contribute to mitochondrial dysfunction and insulin resistance. We have explored the O-GlcNAcylation of mitochondrial proteins from myoblast cells under basal (4mM) and high glucose (30mM) conditions using a combination of proteomic approaches. Furthermore, we have assessed the accompanied changes in the phosphorylation of mitochondrial proteins. We report that a number of mitochondrial proteins are O-GlcNAcylated under basal condition which is altered under high glucose condition. In addition, we report that exposure to high glucose not only changes the O-GlcNAcylation of mitochondrial proteins but also changes their phosphorylation profiles. The dynamic and complex interplay between O-GlcNAcylation and phosphorylation of mitochondrial proteins was further validated by immunoblot analysis of HSP60, prohibitin, and voltage-dependent anion channel 1 as candidate proteins. O-GlcNAcylation of mitochondrial proteins may play a role in normal functioning of mitochondria. High glucose induced changes in O-GlcNAcylation and phosphorylation of mitochondrial proteins may be associated with mitochondrial dysfunction and insulin resistance., (Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.)

Prohibitin-1 (PHB, also known as PHB1), a member of the Band-7 family of proteins, is highly conserved evolutionarily, widely expressed, and present in different cellular compartments. Genetic studies with different organism models have provided strong evidence for an important biological role of PHB in mitochondrial function, cell proliferation, and development. Recent discoveries regarding the involvement of PHB in phophatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and transforming growth factor-β (TGF-β)/signal transducers and activators of transcription signaling pathways, and earlier reports on the interaction of PHB with Raf and its critical role in Ras/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling opened up the possibility that PHB has functions outside of the mitochondria (extramitochondrial) and may be a multifunctional protein. The PI3K/Akt and Ras/MAPK/ERK signaling cascades are versatile signaling processes that diverge from the same receptor tyrosine kinase root, and are involved in cell metabolism, proliferation, and development. Here, we review the emerging role of PHB and its post-translational modifications in signal transduction pathways, especially in PI3K/Akt and Ras/MAPK/ERK signaling. A recent discovery of opposing effects of PHB on longevity under different metabolic states and its potential connection with insulin/insulin-like growth factor-I signaling is also discussed., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Plasma membrane translocation of specific cytosolic proteins plays an important role in cell signaling pathways. We have recently shown that prohibitin (PHB) , a protein present in the plasma membranes of various cell types, interacts with Eps 15 homology domain protein 2 (EHD2), a lipid raft protein. However, the mechanism involved in membrane translocation of PHB is not known.We report that PHB undergoes palmitoylation at cysteine 69 (Cys69), and that this palmitoylation is required for PHB's membrane translocation. Furthermore, we demonstrate that membrane translocation of PHB facilitates tyrosine phosphorylation and its interaction with EHD2. Thus, the palmitoylation and membrane translocation of PHB and its interaction with EHD2 may play a role in cell signaling.

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity in various cells of different origins. Here, we present evidence that the underlying molecular mechanisms involve both programmed cell death I (PCD I, apoptosis) and PCD II (autophagy)-like death. Treatment of cells with S100A8/A9 caused the increase of Beclin-1 expression as well as Atg12-Atg5 formation. S100A8/A9-induced cell death was partially inhibited by the specific PI3-kinase class III inhibitor, 3-methyladenine (3-MA), and by the vacuole H(+)-ATPase inhibitor, bafilomycin-A1 (Baf-A1). S100A8/A9 provoked the translocation of BNIP3, a BH3 only pro-apoptotic Bcl2 family member, to mitochondria. Consistent with this finding, DeltaTM-BNIP3 overexpression partially inhibited S100A8/A9-induced cell death, decreased reactive oxygen species (ROS) generation, and partially protected against the decrease in mitochondrial transmembrane potential in S100A8/A9-treated cells. In addition, either DeltaTM-BNIP3 overexpression or N-acetyl-L-cysteine co-treatment decreased lysosomal activation in cells treated with S100A8/A9. Our data indicate that S100A8/A9-promoted cell death occurs through the cross-talk of mitochondria and lysosomes via ROS and the process involves BNIP3.

Ubiquitination is a highly ordered multistep enzymatic process, carried out by a well-defined systematic ubiquitin pathway, which is required for maintaining appropriate levels and functional activities of various cellular proteins. Targeted regulation of proteins by ubiquitin pathway controls numerous cellular processes including cell proliferation, signal transduction, apoptosis, transcriptional regulation, receptor modulation as well as endocytosis. Significant progress has been made in recent years in not only understanding the structure, function and important regulatory roles of ubiquitin network but also the alterations of ubiquitin pathway in various human diseases including cancer. Based on the progress made, it is now possible to target specifically various components involved in the ubiquitin pathway such as E3 ubiquitin ligases, deubiquitinases and proteosome for potential anticancer therapies. Here we review the potential drug targets available in the ubiquitin system and the small molecule inhibitors that can target these components in the pathway, which can be developed into novel anticancer therapeutics in the near future.

Mitochondrial protein prohibitin (PHB) is known to associate with the plasma membrane of various cell types. However, biological function of plasma membrane associated PHB is not known. Recently we have shown that PHB undergoes tyrosine phosphorylation in response to insulin and here we report that PHB interacts with phosphatidylinositol 3,4,5-triphosphate (PIP3). Furthermore, we demonstrate that over expression of PHB attenuates insulin signaling downstream of phosphatidylinositol 3 (PI3) kinase. This effect was not observed with over expression of tyrosine phosphorylation site mutant-PHB suggesting a role for tyrosine phosphorylation of PHB in this process. Interestingly phosphorylation of PHB by Akt attenuates its interaction with PIP3 and enhances insulin signaling. Thus, phosphorylation of PHB and its interaction with PIP3 may be a part of the regulatory mechanisms that is involved in the modulation of insulin signaling. We speculate that phosphorylation of PHB may serve as a general mechanism in the regulation of PI3 kinase signaling including growth factors and immune receptor signaling.

Prohibitin (PHB or PHB1) is an evolutionarily conserved ubiquitously expressed multifunctional protein and is present in various cellular compartments. Phosphorylation of PHB has been suggested as one of the potential mechanisms in the regulation of its various functions however exact sites of phosphorylation remain to be determined. To better understand the functional relevance of phosphorylation of PHB, we have explored the potential sites of phosphorylation using combination of approaches including phosphoamino specific immunoblotting, proteolysis, two-dimensional gel electrophoresis, phosphoamino acid analysis and site-directed mutagenesis techniques and report that tyrosine 114 (Tyr 114) in PHB is phosphorylated in response to insulin stimulation. In addition, using active insulin receptor (IR) and synthetic biotinylated PHB peptide (PHB(107-121)) we have shown that IR also phosphorylates Tyr 114 in an in vitro kinase assay. Phosphorylation of PHB at Tyr 114 was confirmed by immunoblotting using anti-phosphoTyr 114 specific antibody. Furthermore, we demonstrate that SH2 domain containing tyrosine phosphatase-1 (Shp1) co-immunoprecipitate with PHB antiserum after insulin induced phosphorylation of PHB. Biotinylated-PHB(107-121) peptide phosphorylated at Tyr 114 was also able to pull down Shp1 in pull down assays. Non-phosphorylated PHB(107-121) peptide, corresponding PHB2(121-135) peptide and Tyr114Phe mutant-PHB fail to pull down Shp1. In summary, we have identified Tyr 114 in PHB as an important site of phosphorylation and phosphorylation at this residue creates a binding site for Shp1 both in vivo and in vitro.

The inactivation of programmed cell death has profound effects not only on the development but also on the overall integrity of multicellular organisms. Beside developmental abnormalities, it may lead to tumorigenesis, autoimmunity, and other serious health problems. Deregulated apoptosis may also be the leading cause of cancer therapy chemoresistance. Caspase family of cysteinyl-proteases plays the key role in the initiation and execution of programmed cell death. This review gives an overview of the role of caspases, their natural modulators like IAPs, FLIPs, and Smac/Diablo in apoptosis and upon inactivation, and also in cancer development. Besides describing the basic mechanisms governing programmed cell death, a large part of this review is dedicated to previous studies that were focused on screening tumours for mutations within caspase genes as well as their regulators. The last part of this review discusses several emerging treatments that involve modulation of caspases and their regulators. Thus, we also highlight caspase cascade modulating experimental anticancer drugs like cFLIP-antagonist CDDO-Me; cIAP1 antagonists OSU-03012 and ME-BS; and XIAP small molecule antagonists 1396-11, 1396-12, 1396-28, triptolide, AEG35156, survivin/Hsp90 antagonist shephedrin, and some of the direct activators of procaspase-3.

Prohibitin (PHB or PHB1) is an evolutionarily conserved, multifunctional protein which is present in various cellular compartments including the plasma membrane. However, mechanisms involved in various functions of PHB are not fully explored yet. Here we report for the first time that PHB interacts with O-linked beta-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and is O-GlcNAc modified; and also undergoes tyrosine phosphorylation in response to insulin. Tyrosine 114 (Tyr114) and tyrosine 259 (Tyr259) in PHB are in the close proximity of potential O-GlcNAc sites serine 121 (Ser121) and threonine 258 (Thr258) respectively. Substitution of Tyr114 and Tyr259 residues in PHB with phenylalanine by site-directed mutagenesis results in reduced tyrosine phosphorylation as well as reduced O-GlcNAc modification of PHB. Surprisingly, this also resulted in enhanced tyrosine phosphorylation and activity of OGT. This is attributed to the presence of similar tyrosine motifs in PHB and OGT. Substitution of Ser121 and Thr258 with alanine and isoleucine respectively resulted in attenuation of O-GlcNAc modification and increased tyrosine phosphorylation of PHB suggesting an association between these two dynamic modifications. Sequence analysis of O-GlcNAc modified proteins having known O-GlcNAc modification site(s) or known tyrosine phosphorylation site(s) revealed a strong potential association between these two posttranslational modifications in various proteins. We speculate that O-GlcNAc modification and tyrosine phosphorylation of PHB play an important role in tyrosine kinase signaling pathways including insulin, growth factors and immune receptors signaling. In addition, we propose that O-GlcNAc modification and tyrosine phosphorylation is a novel previously unidentified binary switch which may provide new mechanistic insights into cell signaling pathways and is open for direct experimental examination.

Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.