Patent Application
20240216336
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The field of the invention relates to methods for treating immunodeficiency diseases or disorders in subjects in need thereof. In particular, the field of the invention relates to methods for the treatment of immunodeficiency diseases or disorders in subjects in need thereof by administering to the subject an effective amount of a therapeutic agent that disassociates hexokinase 1 (HK1) from the outer membrane of mitochondria and into the cytosol of macrophage cells in the subject and induces elevated production of inflammatory cytokines including IL-1β, TNFα, and IL-6. The field of the invention also relates to methods for inducing elevated production of inflammatory cytokines including IL-1β, TNFα, and IL-6 in a subject in need thereof.
Immunosuppressive disorders are a major problem in the United States and the number of immunocompromised patients is expected to increase. The spectrum of primary and secondary immunodeficiency disorders is vast and expanding. Medical advances such as organ transplantation and chemotherapy have prolonged the lives of millions, but often at the expense of severe immunodeficiency in the patients. Additionally, primary immunodeficiency disorders encompass a plethora of disease involving the innate and adaptive immune system that often affects children and can lead to premature death in this population. The underlying problem in many of these disorders is an ineffective ability to mount an inflammatory response. This inability to properly active innate and adaptive inflammatory response leaves the patient susceptible to opportunistic infections, which can be fatal. Therefore, it is imperative to find novel therapies to help restore normal inflammatory function in these patients.
In the present application, the inventors describe for the first time that clotrimazole can activate the innate inflammatory response of macrophages in vitro. Clotrimazole was first used as an anti-fungal agent, however, it has been used for a variety of diseases including sickle cell anemia, malaria, Chagas disease, and cancer. Here it is shown that clotrimazole can induce elevated production of inflammatory cytokines including IL-1β, TNFα, and IL-6. These inflammatory cytokines are critical mediators for mounting an immunologic defense against foreign pathogens. Therefore, for the first time clotrimazole is used as a novel therapeutic agent that can be used for boosting the inflammatory response of immunosuppressed patients.
Disclosed are methods for treating immunodeficiency diseases or disorders in subjects in need thereof. The disclosed methods of treatment may include methods for treating immunodeficiency diseases or disorders in subjects in need thereof by administering to the subject an effective amount of a therapeutic agent that results in dissociation of hexokinase 1 (HK1) from the outer membrane of mitochondria and into the cytosol of macrophage cells in the subject. In particular, the disclosed methods may include methods for elevating production of inflammatory cytokines in subjects in need thereof by administering to the subject an effective amount of a therapeutic agent that results in dissociation of hexokinase 1 (HK1) from the outer membrane of mitochondria and into the cytosol of macrophage cells in the subject.
Immunodeficiency disease or disorders treated by the disclosed methods may include, but are not limited to, HIV infection, ataxia-telangiectasia, Chediak-Higashi syndrome, combined immunodeficiency disease, complement deficiencies, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects, panhypogammaglubulinemia, Bruton's disease, congenital agammaglobulinemia, selective deficiency of IgA, or Wiskott-Aldrich syndrome. The immunodeficiency disease or disorder treated by the disclosed methods may also be the result of a family history of primary immunodeficiency, the result of spleen removal, the result of cancer, or the result of liver cirrhosis in the subject in need thereof.
The therapeutic agents that are administered to a subject in the disclosed methods may induce elevated production of IL-1β, TNFα, and IL-6, for example by macrophage cells. The therapeutic agents that are administered to a subject in the disclosed methods may inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, for example in macrophage cells. The therapeutic agents that are administered to a subject in the disclosed methods may increase the activity of the pentose phosphate pathway (PPP), for example in macrophage cells. The therapeutic agents that are administered to a subject in the disclosed methods may induce a hyper-inflammatory response in the subject. Suitable therapeutic agents for use in the disclosed methods that result in the dissociation of HK1 from the outer membrane of mitochondria and into the cytosol may include, but are not limited to, clotrimazole, bifonazole, econazole, ketoconazole, miconazole, and tiocinazole.
The disclosed methods of treatment may include methods for treating immunodeficiency diseases or disorders in subjects in need thereof by administering to the subject an effective amount of a therapeutic agent that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The disclosed methods may include methods for elevating production of inflammatory cytokines in subjects in need thereof by administering to the subject an effective amount of a therapeutic agent that inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Suitable therapeutic agents that inhibit the activity of GAPDH may include, but are not limited to CGP 3466 maleate (CAS 200189-97-5), heptelidic acid, deprenyl, and dihydromanumycin A.
(J) GAPDH activity normalized to total protein in HepG2 cells between FLHK1 and TrHK1 normalized to EV (n=6 replicates per condition for, unpaired t-test, mean±SD). (K) Heatmap of steady-state metabolomics performed on whole mouse brain normalized to total-iron-content in the cells (heatmap generated with MetaboAnalyst).
The present invention is described herein using several definitions, as set forth below and throughout the application.
Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “an inhibitor of tumor cell aggregation” should be interpreted to mean “one or more inhibitors of tumor cell aggregation.”
As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of these terms which are not clear to persons of ordinary skill in the art given the context in which they are used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising” in that these latter terms are “open” transitional terms that do not limit claims only to the recited elements succeeding these transitional terms. The term “consisting of,” while encompassed by the term “comprising,” should be interpreted as a “closed” transitional term that limits claims only to the recited elements succeeding this transitional term. The term “consisting essentially of,” while encompassed by the term “comprising,” should be interpreted as a “partially closed” transitional term which permits additional elements succeeding this transitional term, but only if those additional elements do not materially affect the basic and novel characteristics of the claim.
As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment, for example, treatment by include administering an effective amount of one or more therapeutic agents that results in dissociation of hexokinase 1 (HK1) from the outer membrane of the mitochondria and into the cytosol of macrophage cells.
A “subject in need of treatment” or a “subject in need” may include a subject having or at risk for developing an immunodeficiency disease or disorder. In particular, a subject in need of treatment may include a subject having or at risk for developing an immunodeficiency disease or disorder selected from HIV infection, ataxia-telangiectasia, Chediak-Higashi syndrome, combined immunodeficiency disease, complement deficiencies, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects, panhypogammaglubulinemia, Bruton's disease, congenital agammaglobulinemia, selective deficiency of IgA, or Wiskott-Aldrich syndrome. A subject in need of treatment may also include a subject having or at risk for developing an immunodeficiency disease or disorder which is the result of a family history of primary immunodeficiency. A subject in need of treatment may also include a subject having or at risk for developing an immunodeficiency disease or disorder which is the result of spleen removal in a subject. A subject in need of treatment may also include a subject having or at risk for developing an immunodeficiency disease or disorder which is the result of cancer or liver cirrhosis in a subject. A subject in need a treatment may include a subject in need of elevated production of inflammatory cytokines including IL-1β, TNFα, and IL-6, and/or a subject that has a disease or disorder that is treated by elevated production of inflammatory cytokines including IL-1β, TNFα, and IL-6.
As used herein, the phrase “effective amount” shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of patients in need of such treatment. An effective amount of a drug that is administered to a particular patient in a particular instance will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art.
In the disclosed methods, a subject in need thereof may be administered an effective amount of a therapeutic agent that results in results in dissociation of hexokinase 1 (HK1) from the outer membrane of mitochondria and into the cytosol of macrophage cells in a subject. Suitable therapeutic agents that may be effective for dissociating hexokinase 1 (HK1) from the outer membrane of the mitochondria into the cytosol of macrophage cells in a subject include clotrimazole, bifonazole, econazole, ketoconazole, miconazole, and tioconazole.
In the disclosed methods, a subject in need thereof may be administered an effective amount of a therapeutic agent that inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (GADPH). As disclosed herein, the term “inhibiting” may include blocking enzyme activity and/or reducing enzyme activity. Suitable therapeutic agents that may inhibit the activity of glyceraldehyde-3-phosphate dehydrogenase (GADPH) in a subject include CGP 3466 maleate (CAS 200189-97-5), heptelidic acid, deprenyl, and dihydromanumycin A.
The disclosed therapeutic methods may be effective for elevating production of inflammatory cytokines in a subject. As disclosed herein, the term “elevating” may include increasing production and/or concentration of inflammatory cytokines in macrophage cells. Inflammatory cytokines include IL-1β, TNFα, and IL-6.
In the disclosed methods, a subject in need thereof may be administered an effective amount of a therapeutic agent that results in results in elevating production of inflammatory cytokines in subjects in need thereof, such as IL-1β, TNFα, and IL-6. Suitable therapeutic agents that may be effective for elevating production of inflammatory cytokines in subjects in need thereof include clotrimazole, bifonazole, econazole, ketoconazole, miconazole, and tioconazole. Suitable therapeutic agents that may be effective for elevating production of inflammatory cytokines in subjects in need thereof also may include CGP 3466 maleate (CAS 200189-97-5), heptelidic acid, deprenyl, and dihydromanumycin A.
The disclosed subject matter relates to methods for treating immunodeficiency diseases or disorders in subjects in need thereof. Suitable subjects for the disclosed methods may include subjects having an immunodeficiency disease or disorder. Immunodeficiency diseases or disorders may include, but are not limited to, an HIV infection, ataxia-telangiectasia, Chediak-Higashi syndrome, combined immunodeficiency disease, complement deficiencies, DiGeorge syndrome, hypogammaglobulinemia, Job syndrome, leukocyte adhesion defects, panhypogammaglubulinemia, Bruton's disease, congenital agammaglobulinemia, selective deficiency of IgA, or Wiskott-Aldrich syndrome. Suitable subjects for the disclosed methods also may include subjects having an immunodeficiency disease or disorder which is the result of a family history of primary immunodeficiency. Suitable subjects for the disclosed methods may also include subjects having an immunodeficiency disease or disorder which is the result of spleen removal in a subject. Suitable subjects for the disclosed methods may also include subjects having an immunodeficiency disease or disorder which is the result of cancer or liver cirrhosis in a subject.
In the disclosed methods, a subject in need thereof may be administered an effective amount of a therapeutic agent that results in dissociation of hexokinase 1 (HK1) from the outer membrane of mitochondria and into the cytosol of macrophage cells in the subject. Suitable therapeutic agents may include, but are not limited to, clotrimazole, bifonazole, econazole, ketoconazole, miconazole, and ticonazole. In particular embodiments, the therapeutic agent is clotrimazole.
In some embodiments of the disclosed methods, the effective amount of the therapeutic agent (e.g., clotrimazole) may induce elevated production of inflammatory cytokines by the macrophage cells. In particular embodiments, the inflammatory cytokines are of IL-1β, TNFα, and IL-6.
In some embodiments, the effective amount of the therapeutic agent (e.g., clotrimazole) may inhibit GAPDH activity in macrophage cells. In some embodiments, the effective amount of the therapeutic agent (e.g., clotrimazole) results in an increase of activity of the pentose phosphate pathway (PPP) in macrophage cells. In some embodiments, the effective amount of the therapeutic agent clotrimazole, induces a hyper-inflammatory response in a subject, which may include but is not limited to an increase in production of inflammatory cytokines such as IL-1β, TNFα, and IL-6.
In the disclosed methods, a subject in need thereof may be administered an effective amount of a therapeutic agent that inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Suitable therapeutic agents that inhibit GAPDH activity may include, but are not limited to, CGP 3466 maleate (CAS 200189-97-5), heptelidic acid, deprenyl, and dihydromanumycin A.
The disclosed subject matter also includes methods for elevating production of inflammatory cytokines in a subject in need thereof are disclosed herein. In the disclosed methods, a subject in need thereof for elevating production of inflammatory cytokines may be administered an effective amount of a therapeutic agent that results in dissociation of hexokinase 1 (HK1) from the outer membrane of mitochondria and into the cytosol of macrophage cells in the subject. In some embodiments, the inflammatory cytokines are IL-1β, TNFα, and IL-6. Suitable therapeutic agents may include but are not limited to clotrimazole, bifonazole, econazole, ketoconazole, miconazole, and ticonazole. In particular embodiments, the therapeutic agent is clotrimazole.
In the disclosed methods, a subject in need thereof for elevating production of inflammatory cytokines may be administered an effective amount of a therapeutic agent that inhibits the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Suitable therapeutic agents may include but are not limited to CGP 3466 maleate (CAS 200189-97-5), heptelidic acid, deprenyl, and dihydromanumycin A.
The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.
Hexokinases (HKs) represent the first committed step in glucose utilization by catalyzing the phosphorylation of glucose to glucose-6-phosphate (G6P) which sequesters glucose in the cell and commits it to various downstream metabolic pathways (Gould and Holman, 1993; Bell et al., 1993; Mueckler, 1994; Printz et al., 1997). G6P can enter glycolysis for energy production, the pentose phosphate pathway (PPP) for anabolic intermediates, the hexosamine biosynthesis pathway (HBP) for protein glycosylation, or be converted to glucose-1-phosphate for glycogen synthesis (Adeva-Andany et al., 2016; Puleston et al., 2017). In mammals, five HK isozymes (HK1, HK2, HK3, glucokinase (GCK), and hexokinase domain-containing 1 (HKDC1)) have been identified, each with distinct patterns of tissue expression, subcellular localization, enzyme kinetics, and substrate specificities (Middleton, 1990; Ureta, 1982; Wilson, 2003). The molecular weights of HK1, 2, 3, and HKDC1 are ˜100 kDa, whereas GCK and yeast HKs have a molecular weight of 50 kDa. The protein and gene structure of the 100 kDa enzymes suggest that they evolved from gene duplication and fusion of an ancestral yeast-like 50 kDa enzyme (Cirdenas et al., 1998; Printz et al., 1997). Upon the duplication process, the N-terminal half of HK1 and HK3 became enzymatically inactive, whereas HK2 maintained activity in both of its catalytic domains (Ardehali et al., 1996).
HK1 and HK2 also contain an N-terminal, 21-amino-acid hydrophobic sequence (Rose and Warms, 1967; Sui and Wilson, 1997) that enables outer mitochondrial membrane (OMM) binding, possibly through its interaction with the family of mitochondrial bound voltage dependent anion channel (VDAC) proteins (Fiek et al., 1982; Linden et al., 1982; Aflalo and Azoulay, 1998; Azoulay-Zohar et al., 2004). This sequence, termed the mitochondrial binding domain (MBD), is encoded in exon 1, while exons 2 through half of exon 10 encode for the N-terminal domain and the remaining part of exon 10 through exon 18 encode for the C-terminal domain (Printz et al., 1995, 1993). Notably, HK1 and HK2 differ in their affinity for mitochondrial localization (Calmettes et al., 2013; John et al., 2011), with HK1 predominantly bound to the OMM, while HK2 is in a dynamic balance between the mitochondria and cytosol that is regulated, in part, by insulin signaling (Pastorino et al., 2005; Roberts et al., 2013). HK3 and GCK lack a corresponding N-terminal sequence and as a consequence are predominantly cytoplasmic (Wilson, 1995; Cirdenas et al., 1998), although some reports suggest that HK3 may bind to the nuclear envelope (Preller and Wilson, 1992).
Metabolic adaptations are inextricably linked to the immediate host defense against foreign pathogens (Pearce and Pearce, 2013; Sancho et al., 2017; Stienstra et al., 2017). In the setting of innate inflammatory activation, there is a robust increase in glycolysis even in the setting of abundant oxygen, a metabolic program shared by rapidly proliferating cells called aerobic glycolysis (Cheng et al., 2014; Everts et al., 2014; Garaude et al., 2016; O'Neill and Pearce, 2016; Pavlova and Thompson, 2016; Andrejeva and Rathmell, 2017). In addition to elevated glycolytic rate, there is increased glucose flux into the PPP and other ancillary metabolic pathways in immune cells following toll-like receptor (TLR) stimulation (Puleston et al., 2017; Hughes and O'Neill, 2018). In macrophages, HK1 mRNA is highly expressed after pro-inflammatory stimulation (Nishizawa et al., 2014), and HK1-dependent glycolysis is important for proper inflammasome activation in M1 macrophages (Moon et al., 2015). Moreover, HK1 has also been shown to facilitate metabolite flux into the mitochondria in CD8+ cells (Bantug et al., 2018). HK2 is also shown to play an important role in inflammasome activation (Wolf et al., 2016), and in viral-mediated inflammation (Zhang et al., 2019). These results indicate a major role for glucose metabolism through HK1 in the activation of inflammatory cells.
Despite extensive research on glucose metabolism, it remains unclear what factor(s) decide the fate of glucose and regulate the entrance of G6P into a specific pathway. Elucidating this regulation will be important for developing rational therapeutic approaches to inflammatory diseases, cancer, and a host of other immune system related pathologies. Here, we demonstrate that the binding of HK1 to the mitochondria determines whether the product of the enzyme (G6P) is catabolized through glycolysis or shunted through PPP. We demonstrate that constitutive dislocation of HK1 to the cytoplasm shifts G6P entry into the PPP, resulting in higher cytokine production and exaggerated inflammatory response to endotoxemia. The mechanism for the altered G6P metabolism by HK1 cellular distribution is through cytosolic HK1 interaction with S100A8/9, which induces S-nitrosylation of GAPDH and its subsequent inactivation. Therefore, HK1 functions as an important metabolic switch between catabolic and anabolic metabolism through its subcellular localization.
HK1 contains a 21-amino acid hydrophobic N-terminal domain that confers outer-mitochondrial membrane (OMM) binding. To determine the cellular consequences of HK1 mitochondrial binding, we generated GFP-tagged constructs of full-length HK1 (FLHK1) and truncated HK1 (TrHK1) that lack the MBD, along with an empty vector (EV) control that lacks an insert (
To determine the physiologic function of the HK1 MBD, we generated a mouse model lacking the MBD of the endogenous HK1. The approach to remove the MBD is depicted in
Immunofluorescence (IF) studies in bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) isolated from WT and ΔE1HK1 mice also confirmed lack of HK1 localization to the mitochondria (
Since there was no difference in glucose phosphorylation with HK1 MBD deletion, we then assessed whether glucose metabolism was altered by the removal of the HK1 MBD. HepG2 cells expressing TrHK1 displayed reduced glycolysis, as assessed by extracellular lactate production and extracellular acidification rate (ECAR) (
We also performed 13C6-glucose labeling metabolomics in HepG2 cells with FLHK1 or TrHK1 overexpression and assessed similar metabolic pathways (
Glycolysis serves a vital role in the initiation and maintenance of proper effector function of activated macrophages (Rodríguez-Prados et al., 2010; Pearce and Pearce, 2013; Everts et al., 2014). PPP metabolism has been shown to be upregulated in LPS activated macrophages and is necessary for proper inflammatory activation (Haschemi et al., 2012). Overexpression of G6PD, the rate-limiting enzyme of the oxidative-PPP, in macrophages was shown to enhance pro-inflammatory cytokine production (Ham et al., 2013). Furthermore, the non-oxidative branch of the PPP was shown to be highly upregulated in LPS activated macrophages, which is thought to be necessary for providing de novo nucleotides to support their characteristic transcriptional response (Martinez et al., 2006; Nagy and Haschemi, 2015). Given the metabolic alterations seen with disruption of HK1 mitochondrial binding, we next assessed the consequence of these changes on the effector function of LPS-activated macrophages. We noted a significant increase in inflammatory cytokines IL-1β, IL-6 and TNFα mRNA expression in BMDMs from ΔE1HK1 mice compared to WT mice in response to LPS (
PMs isolated from ΔE1HK1 mice also showed increased IL-1β, IL-6 and TNFα mRNA expression relative to WT cells when stimulated with LPS, similar to BMDMs (
The PPP is upregulated in pro-inflammatory (M1) macrophages and neutrophils, and reduction of PPP metabolites reduces M1 macrophage cytokine production (Nagy and Haschemi, 2015; Baardman et al., 2018). Thus, to determine whether the hyper-inflammatory response seen in ΔE1HK1 mice is dependent on the increased PPP metabolism, we used two methods to inhibit the PPP in BMDMs from these mice: 1) 6-aminonicotinamide (6AN), which is metabolized into an NADP+ analog and competitively inhibits NADPH-producing enzymes 6-phosphogluconate dehydrogenase (PGD) and glucose 6-phosphate dehydrogenase (G6PD), and 2) oxythiamine (OT), a thiamine antagonist, which suppresses the non-oxidative synthesis of ribose by inhibiting the transketolase (TKT) enzyme (
GAPDH Activity is Attenuated in Macrophages with HK1 Mitochondrial Detachment
We next studied the mechanism by which cytosolic HK1 reduces glycolysis and shifts G6P towards the PPP. Analysis of the 13C6-glucose labeling metabolomics indicated an increase in metabolites in upper glycolysis (i.e., metabolites upstream of the GAPDH-mediated step in glycolysis) and a reduction in metabolites in lower glycolysis (i.e., downstream of GAPDH), suggesting a block in glycolysis at the level of GAPDH. As expected PMs and BMDMs from ΔE1HK1 mice displayed significantly decreased GAPDH activity at baseline and with LPS treatment (
We also performed steady-state metabolomics in HepG2 cells with TrHK1 overexpression and observed a similar metabolic block at the level of GAPDH (
We next assessed the effects of acute dislocation of HK1 from the mitochondria (as opposed to constitutive dislocation of the protein, as occurs in the ΔE1HK1 mice) on GAPDH activity. Clotrimazole (CLT), an antifungal drug, is known to dislocate HKs from the mitochondria (Huang et al., 2002; Shoshan-Barmatz et al., 2010; Sen et al., 2015). As expected, CLT caused a significant increase in HK1 dislocation from the mitochondria in WT BMDMs (
We next studied the mechanism of GAPDH inactivation with HK1 mitochondrial dissociation. We hypothesized that cytosolic HK1 binds to a distinct set of proteins compared to the mitochondrial bound HK1. To test this, we performed an unbiased immunoprecipitation (IP) experiment followed by mass spectrometry (MS) in HepG2 cells with overexpression of FLHK1 and TrHK1. Proteomic analysis yielded 611 unique spectra which corresponded to 175 identified proteins (
We then confirmed this finding in BMDMs from ΔE1HK1 mice and found S100A8 as a binding partner of cytosolic HK1 in these cells (
Diabetes and Aging are Associated with HK1 Mitochondrial Dislocation and Increased Cytokine Production
Our data thus far indicates that constitutive or acute HK1 dislocation causes an increase in cytokine production through inhibition of GAPDH, which alters glycolysis and subsequently increases PPP metabolism. We next studied whether subcellular localization of HK1 is altered in conditions of chronic low-grade inflammation, such as diabetes and aging. To generate diabetic mice, we fed 4-week-old mice a high fat diet (HFD) consisting of 60% fat, 20% protein, and 20% carbohydrates. Mice given HFD for 27 weeks displayed impaired glucose tolerance and increased body weight as compared to mice fed normal chow (NC) (
Here, we show that HK1 mitochondrial dissociation produces a metabolic block at the level of GAPDH, which increases PPP metabolism. This feature of HK1 regulation is dependent on its localization between the cytosol and mitochondria and is independent of its enzymatic activity, since removal of HK1 MBD does not change its enzymatic function. We also demonstrate that the increased PPP metabolism has functional consequences, as dislocation of HK1 to the cytoplasm results in higher cytokine production and exaggerated inflammatory response to endotoxemia in vivo. Additionally, we show that the mechanism for the altered G6P metabolism by HK1 cellular distribution is through increased GAPDH S-nitrosylation and subsequent attenuation of its enzymatic activity. Our data suggest that cytosolic HK1 binds to S100A8/A9, leading to S-nitrosylation of GAPDH through iNOS. Furthermore, we find that inhibition of iNOS is sufficient to reverse the metabolic alterations and elevated cytokine production seen with mitochondrial dissociation of HK1. Therefore, HK1 subcellular localization is a critical modulator of glycolysis, and regulates the inflammatory response in macrophages (
The hydrophobic N-terminal domain of HK1 and HK2 encoded by exon 1 allows the proteins to bind to the mitochondria (John et al., 2011; Wilson, 2003). We previously showed that the mitochondrial binding of HK1 and HK2 are needed for their protective effects against cell death (Sun et al., 2008). Other reports have suggested that the binding of HK1 and HK2 to the mitochondrial allows preferential access of these enzymes to mitochondrial-produced ATP that is transported through VDAC, in addition to effective delivery of ADP that is produced by glucose phosphorylation to ANT for transport to mitochondrial matrix (BeltrandelRio and Wilson, 1992; Golshani-Hebroni and Bessman, 1997; Rosano et al., 1999). However, the Km for ATP of these enzymes is significantly lower than cytosolic ATP (Zeng et al., 1996; Rosano et al., 1999), making the validity of this hypothesis dubious. Thus, the physiological significance of HK1 binding to the mitochondria is not clear. G6P can shuttle through different pathways (Adeva-Andany et al., 2016), and despite extensive work on glucose metabolism, there is a paucity in our understanding of how the metabolic fate of G6P is determined. Here, we identify a mechanism by which the localization of HK1, independent of its enzymatic activity, regulates the metabolic fate of its enzymatic product, G6P, and increases its shunting into the PPP. These data highlight how subcellular localization of a metabolic enzyme determines the fate of its product.
We also showed that the mechanism by which cytosolic HK1 increases PPP in macrophages is through GAPDH nitrosylation and enzymatic inhibition. A role for GAPDH nitrosylation in inflammation has been previously reported. Nitrosylation of GAPDH has been previously shown to increase inflammation in macrophages (Jia et al., 2014; Padgett and Whorton, 1995), and GAPDH inhibitors cause an increase in IL-1β secretion by macrophages (Sanman et al., 2016). Additionally, a role for increased PPP metabolites in inflammation has been suggested before (Baardman et al., 2018; Haschemi et al., 2012). Our results provide a biological context and mechanism for GAPDH regulation and links these steps to HK1 mitochondrial binding. We showed that HK1 dislocation from the mitochondria leads to its association with S100A8/A9, nitrosylation of GAPDH and its subsequent inhibition. GAPDH is a critical step in glycolysis in that the substrates above this step can enter the PPP, and thus its inhibition would block glycolysis, while allowing shuttling of upstream intermediates into PPP. We suspect that this system was developed in mammalian cells to serve as a means of regulating the metabolic fate of G6P into ancillary pathways off glycolysis, such as the PPP. Since the metabolic block at GAPDH increases metabolite levels above this point, it would be interesting to investigate how other metabolic branch points of G6P (i.e., glycogenesis and hexosamine pathway) are affected in other contexts.
In light of our findings, it is now important to determine both physiological and pharmacological factors that modulate HK1 binding to the mitochondria and its release into the cytoplasm. HK1 dislocation from the mitochondria has been reported to occur in response to a number of pathways and processes, including senescence, ROS, CLT, inhibition of AKT, and activation of GSK (Gardiner et al., 2007; Saraiva et al., 2010; John et al., 2011; Fouquerel et al., 2014; Sen et al., 2015; Hauser et al., 2017; Bantug et al., 2018; Mogilenko et al., 2019). These findings indicate that there is shuttling of HK1 from the mitochondria to the cytoplasm under physiological and pathological conditions. In addition, a synthetic peptide with sequence homology to the N-terminus of HK1 can be used to displace the proteins from the mitochondria (Magri et al., 2017). Thus, this peptide can potentially be used in clinical settings to induce HK1 displacement from mitochondria and increase the inflammatory response in conditions of impaired inflammation, such as immunosuppressive disorders. We also showed that in the setting of certain conditions associated with low-grade inflammation, such as aging and diabetes, there is endogenous HK1 dislocation from the mitochondria, increased production of inflammatory markers, and diminished GAPDH activity with LPS stimulation. Additionally, treatment with an iNOS inhibitor showed a cell-autonomous reversal of the elevated cytokine production observed in DIO PMs along with restoration of GAPDH activity. These results indicate that HK1 mitochondrial binding may mediate the inflammation underlying the pathogenesis of diabetes and aging. Thus, it would be important to devise strategies to maintain HK1 on the mitochondria to reduce or ablate the inflammation (either low-grade or fulminant) that contributes to the negative sequelae of these and other inflammation-associated disorders.
In summary, we have identified a novel mechanism by which the product of the HK enzymatic reaction, G6P, is directed towards the PPP at the expense of a reduction in lower glycolytic intermediates. This metabolic effect is regulated through HK1 mitochondrial interaction and that the mitochondrial dissociation of this enzyme causes an increase in PPP through inhibition of GAPDH. We provide data suggesting this mechanism is mediated through HK1 interaction with S100A8/A9, which increased iNOS activity and subsequent GAPDH nitrosylation and inhibition. These results provide a new mechanism by which the subcellular localization of a glycolytic enzyme regulates its own downstream metabolism to produce an effect on inflammatory cytokine production.
RAW264.7 cells were a gift from Jason A. Wertheim MD, PhD. HepG2 and HEK293T cells were obtained from ATCC. RAW264.7, HEK293T, and HepG2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Corning) supplemented with 10% FBS (Atlanta Biologicals), 2 mM glutamine (HyClone), and 1 mM sodium pyruvate (Corning). RAW264.7 cells were treated with 300 ng/ml LPS for 4-6 hrs with or without CLT (50 μM).
HK1ΔE1HK1/ΔE1HK1 (ΔE1HK1) and HK1WT/WT wild type (WT) control mice were generated by the Northwestern Mutagenesis and Transgenic core. Mouse genetic background is C57BL/6J. Mice were maintained in the barrier facility at Northwestern University under specific pathogen-free conditions in accordance with Federal and University guidelines and protocols approved by Institutional Animal Care and Use Committee (IACUC) with 12 hr light and 12 hr dark cycle, and received normal chow. Male and female mice were used at 8-12 weeks of age. HFD and NC diet mice were maintained on respective diets for 27 weeks. All animal studies were approved by the IACUC at Northwestern University and were performed in accordance with guidelines from the National Institutes of Health.
Peritoneal macrophages were isolated from 8-10-week-old ΔE1HK1 and littermate control WT mice by peritoneal lavage as described previously (Ray and Dittel, 2010). Briefly, mice were euthanized and the peritoneal cavity was injected with PBS supplemented with 5% FBS using a 27g needle. The abdominal wall was gently agitated to dislodge peritoneal cells. Using a fresh 25g needle, the peritoneal lavage (PBS and peritoneal cells) was aspirated and collected. Cells were then centrifuged at 500 g for 10 min and re-suspended in 1 mL of complete RPMI (Corning) supplemented with 10% FBS (Atlanta Biologicals), 2 mM glutamine (HyClone), 1 mM HEPES (Corning) and 1 mM pyruvate (Corning). Stimulation with LPS (Invivogen) was performed the next day. 300 ng/ml of LPS was given to the cells for 4-6 hrs with or without the following drugs; OT (50 μM), 6-AN (1 mM), 1400W (50 μM), or CGP3466 (50 nM).
BMDMs were isolated from ΔE1HK1 and WT mice as previously reported (Rodriguez et al., 2019). Briefly, bone marrow was isolated from the tibia and femur of 8-10-week-old mice by puncturing one end of the femur and tibia with a 27g needle and placing it in a 0.5 ml tube with a hole punched in the bottom. The 0.5 ml tube containing the bone was then placed in a 1.75 ml tube and centrifuged at 5,000×g for 3.5 minutes. Bone marrow was then collected into the 1.75 ml tube and resuspend in 1 ml of RBC lysis buffer (abcam) for 1 minute and then transferred to a 50 ml tube with 4 ml RBC lysis buffer and incubated for 4 minutes. Next, 35 ml of PBS was added and centrifuged at 300×g for 5 min and supernatant was then decanted and cell pellet was re-suspended in complete RPMI with 20 ng/ml mCSF (Preprotech). Cells were counted using a hemocytometer and plated in 10 cm tissue culture plates at a density of 3-million cells/plate. Cells were cultured with 20 ng/mL mCSF to induce differentiation into BMDMs. Media was changed every 2 days and BMDMs were harvested by scraping on day 6 and plated in 12 well plates at a density of 1-million cells/well for mRNA extraction experiments. BMDMs were stimulated in the same manner as peritoneal macrophages (LPS dose for BMDMs was 200 ng/ml).
Lentivirus C-terminal GFP-tag fusion over-expression constructs for EV, FLHK1, and TrHK1 were cloned into the pHIV-Puro vector (generated in our lab) using InFusion-HD cloning method. Lentiviral particles were produced in HEK293T cells co-transfected with pSPAX2 and pMD2.G packaging vectors using standard protocols. To generate the full-length and truncated HK1-GFP fusion plasmids, In-Fusion cloning PCR primers were designed to the first or second exon of the 2754-bp fragment from the EGFP-N3 plasmid containing FLHK1 or TrHK1. To perform the In-Fusion reaction, the PCR primers also contained overhang regions containing XhoI and BamHI-HF restriction enzyme sites and a 15-bp homology portion to the pHIV-Puro vector to facilitate recombination.
RNA was isolated from cells or tissues using RNA-STAT60 (Teltest) followed by chloroform extraction and precipitation. Reverse transcription was carried out using qScript cDNA Synthesis Kit (Quanta Bio). The resulting cDNA was amplified quantitatively using PerfeCTa SYBR Green Mix (Quanta Bio) on a 7500 Fast Real-time PCR System (Applied Biosystems). The relative gene expression was determined using differences in Ct values between gene of interest and house-keeping control genes. Complete list of primers can be found in Key Reagents Table (Table 1).
Cells and tissue were lysed in radio-immunoprecipitation assay (RIPA) buffer supplemented with 1× protease inhibitor (G-Bioscience). Protein concentration in samples was determined using the BCA Protein Quantification Kit (Pierce). Equal amounts of protein were loaded on a tris-glycine polyacrylamide gel (Life Technologies) and transferred to nitrocellulose membrane. After blocking with tris-buffered saline containing 0.05% Tween 20 (DOT Scientific Inc) and 5% BSA, the membrane was incubated with primary antibody against indicated proteins. A complete list of antibodies is included in Key Resources Table (Table 1). For low molecular weight protein S100A8 western blots, 16% tricine protein gels (ThermoFisher) were used and western blots run as previously described (Schägger, 2006). Western blot densitometry was performed using Fiji/ImageJ Gel analyzer macro (Schindelin et al., 2012).
Glass-bottom confocal dishes (35 mm; VWR) were coated with 60 nM fibronectin (Sigma-Aldrich), diluted in 0.1% gelatin overnight. Before plating cells, coated confocal dishes were washed twice with PBS. BMDMs (100,000 cells/well), PMs (100,000 cells/well), HepG2 (50,000 cells/well), or RAW264.7 (50,000 cells/well) cells were plated in their respective growth media (complete DMEM or RPMI) and allowed to settle overnight before immunofluorescence (IF) staining. For fixed cell immunofluorescence (IF), cells were plated in 6-well plates (Corning) containing 15 mm glass coverslips (Fisher-Scientific) coated in gelatin and fibronectin. Prior to fixation, growth media was removed and cells were washed 2× with PBS. Cells were fixed with 1 ml of ice-cold 4% formaldehyde for 10 min at room temperature (4% formaldehyde prepared from 16% stock and diluted in PBS). After fixing the cells, plates were washed with PBS 2× and permeabilized with 0.3% triton-X-100 in PBS for 10 min at room temperature. Then, cells were blocked with 10% FBS/PBS for 1 hour at room temperature and subsequently treated with primary antibody diluted in 10% FBS/PBS solution over-night. The next day, secondary antibody was diluted in 10% FBS/PBS at 1:1000 and incubated for 2 hrs at room temperature. Cells were then washed 3× with PBS and mounted on glass slides (Fisher-Scientific) using ProLong Gold Antifade Mountant with DAPI and imaged using confocal microscope. HK1 antibody diluted at 1:100, ATP Synthase-beta (ATP5B) Monoclonal-Alexa Fluor 555 antibody diluted at (1:200), and MitoTracker Deep Red FM (1 μM). All images were acquired on a Zeiss LSM 510 Meta confocal microscope. Images were quantified using ImageJ.
HepG2 cells with EV, FLHK1, or TrHK1 overexpression were washed with PBS and changed to confocal buffer (25 mM D-glucose, 1.8 mM CaCl2, 2.5 mM KCl, 140 mM NaCl, 2 mM sodium pyruvate, 2 mM glutamine, 20 mM HEPES, pH 7.5, 1 mM MgCl2). Cells were stained with 5 nM TMRE (for mitochondrial stain) for 20 minutes. Cells were then washed with PBS, and fresh confocal buffer was added. TMRE (red channel) and GFP (green channel) signals were analyzed for colocalization using ImageJ Coloc-2 function to determine Pearson correlation coefficient between green and red channel for confocal images.
13C6-Glucose Tracing and Steady-State Metabolomics
Cultured BMDMs or HepG2 cells were treated with 13C6-glucose for 4 hrs±LPS (200 ng/ml) and mass-spectrometry and metabolite identification was performed on 80% methanol & 20% ultrapure water extracted metabolites. Whole brain tissues were harvested from mice and immediately flash frozen in liquid nitrogen until harvested for metabolites using 80% methanol & 20% ultrapure water extraction protocol. Metabolomics services were performed by the Metabolomics Core Facility at Robert H. Lurie Comprehensive Cancer Center of Northwestern University. Samples were analyzed by High-Performance Liquid Chromatography and High-Resolution Mass Spectrometry and Tandem Mass Spectrometry (HPLC-MS/MS). Specifically, system consisted of a Thermo Q-Exactive in line with an electrospray source and an Ultimate3000 (Thermo) series HPLC consisting of a binary pump, degasser, and auto-sampler outfitted with an Xbridge Amide column (Waters; dimensions of 4.6 mm×100 mm and a 3.5 μm particle size). The mobile phase A contained 95% (vol/vol) water, 5% (vol/vol) acetonitrile, 20 mM ammonium hydroxide, 20 mM ammonium acetate, pH=9.0; B was 100% Acetonitrile. The gradient was as following: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1-18 min, 75% A; 18-25 min, 15% A with a flow rate of 400 μL/min. The capillary of the ESI source was set to 275° C., with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative polarity switching mode, an m/z scan range from 70 to 850 was chosen and MS1 data was collected at a resolution of 70,000. The automatic gain control (AGC) target was set at 1×106 and the maximum injection time was 200 ms. The top 5 precursor ions were subsequently fragmented, in a data-dependent manner, using the higher energy collisional dissociation (HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500. The sample volumes of 10 μl were injected. Data acquisition and analysis were carried out by Xcalibur 4.0 software and Tracefinder 2.1 software, respectively (both from Thermo Fisher Scientific).
2-NBDG glucose uptake assay of LPS-activated BMDMs from WT and ΔE1HK1 mice was performed based on previous reports (Alonso-Castro and Salazar-Olivo, 2008). Briefly, cells were cultured overnight in 96 well plates and treated the next day with 300 μM 2NBDG for 1 hr and then quickly washed 3× with PBS. Cells were then re-suspended in RIPA buffer and imaged in a fluorescent plate reader.
BMDMs were stimulated with O5:B55 LPS (Invivogen) for 6 hours with addition of 2 mM ATP (Sigma) for 30 minutes to activate the cleavage of pro-IL-1β (35 kDa) to cleaved-IL-1β (17 kDa). Cell supernatant was collected and analyzed by ELISA according to manufacturer's instructions for IL-1β (DY401), IL-6 (DY406), and TNFα (DY410).
GAPDH immunoprecipitation of HepG2 and BMDM cells was performed and nitrosylated cysteines were replaced with covalent binding of TMT using Pierce S-Nitrosylation western blot kit according to manufactures protocol. Briefly, free cysteines were blocked with MMTS reagent, lysates were treated with iodoTMT/ascorbate to induce replacement of unstable S-NO with stable S-TMT moiety. Western blot was then run on the TMT-replaced lysates and probed with GAPDH (dilution 1:8,000) and anti-TMT (dilution 1:1000).
The day before the assay, the Seahorse cartridge was placed in the XF calibrant and incubated overnight at 37° C. On the day of the assay, cells were seeded into the Seahorse 96-well plate at 15,000 cells/80 μl per well for HepG2 cells or 100,000 cells/80 μl per well for BMDMs. The plates were incubated at RT for 1 hour in glucose free complete DMEM or RPMI without bicarbonate or phenol-red to allow even distribution of cells across the well floor. Before placing the sample plates in the Seahorse XF96 Analyzer, medium volume was adjusted to 175 μl in each well. 11 mM or 25 mM glucose for HepG2 cells or BMDMs respectively, Oligomycin at 2 μM, CCCP at 10 μM, and 2DG at 2 μM each, diluted in DMEM, were injected sequentially into each well including control wells, containing only medium, following the standard Seahorse protocol. For acute injection of LPS, the first port of the drug cartridge was replaced with LPS at 200 ng/ml.
LPS induced sepsis model in mice was approved by Northwestern University Institutional Animal Care and Use Committee. For short term LPS induced cytokine quantification, C57/B16 mice (aged 10-12 weeks) were treated i.p. with or without 1400W (10 mg/kg) for 2 hrs prior to i.p. treatment with ultrapure 05:B55 LPS from Invivogen (15 mg/kg) i.p. for 4 hours. Whole blood samples were harvested via cardiac puncture after mice were euthanized. Cytokine production in serum from whole blood was measured using the Mouse IL-1β (DY401), IL-6 (DY406), and TNFα (DY410) ELISAs from R&D. For survival studies, Crude 05:B55 LPS (Sigma) was administrated i.p. at a sub-lethal dose of 15 mg/kg and mice were monitored over 72 hrs, every 2-4 hrs for survival and signs of deterioration to determine humane endpoints (Shrum et al., 2014).
The following kits were used according to the manufacturer's instructions: Promega NADP/NADPH quantification kit (G9081) and NAD/NADPH quantification kit (G9071); Glyceraldehyde-3-Phosphate Dehydrogenase Activity Assay kit (abcam); ELISAs for IL-1β (DY401), IL-6 (DY410), and TNFα (DY406).
CBC and WBC from Whole Blood
A HEMEVET blood cell analyzer was used on whole blood from mice using mouse standard blood as a control for comparing readouts.
Anti-HK1 magnetic beads IP kit (Sino Biological) was used to IP HK1 from BMDMs. BMDMs were plated in 15 cm culture dishes and lysed using NP40 cell lysis buffer (Sino Biological-provided in kit) and sonicated for 5 pulses for 1 sec each. Lysate was centrifuged at 8,000×g and supernatant was collected in a fresh tube. 50 μL of HK1-magnetic beads was added to afresh 1.7 mL tube and washed with 150 μL 1×TBS (10×TBS: 60.6 g Tris, 87.6 g NaCl, 1M HCl, 7.5 pH) with 0.5% Tween-20 (DOT Scientific Inc) and beads were precipitated with magnetic separator (Sino Biological-provided in kit). 1,000 ug of lysate from ΔE1HK1 and WT BMDMs was added to the precipitated magnetic HK1 beads and incubated overnight at 4° in rotator. Next day, beads were magnetically precipitated and washed 3× with 1×TBST. Bound protein from precipitated beads was eluted using acidity elution buffer (Sino Biological-provided in kit) and western blot analysis was performed.
HepG2 cells were plated to confluency on 15 cm dishes and scrapped in 10-mL PBS and collected into 15 mL conical tube. Cells in PBS were then centrifuged at 500×g for 15 min at 4° C. Supernatant was then aspirated and cell pellet was re-suspended in 300 μL co-IP lysis buffer (100 mM HEPES-pH 7.7, 250 mM KCl, 2 mM MgCl2, 2 mM EDTA, 10% glycerol, 1% digitonin) and then transferred to 1.7 mL tubes. The lysate was then sonicated for five pulses of 1 second each and incubated on ice for 30 minutes. Lysates were then centrifuged at 16,000×g for 15 min and supernatant was collected in fresh 1.7 mL tube. Protein quantification was performed using BCA (Pierce) and 2,000 μg of protein was used for co-IP. Co-IP was performed using GFP-Trap-Agarose beads (Chromotek) according to manufacturer's protocol. Eluted samples were given to the Northwestern Proteomics Core using the 100-minute gradient tandem mass spectrometry Orbitrap.
Lactate quantification adapted from previous work (Gandhi et al., 2009). The lactate reporter system contained 200 μmol/L Amplex red (Molecular Probes), 4 units/mL lactate oxidase (Sigma), and 0.8 units/mL horseradish peroxidase (Sigma) in 50 mmol/L Tris-HCl, pH 7.4, which was deoxygenated with helium to reduce oxidation of Amplex red. The assays were incubated for 30 min, followed by measurement of fluorescence at 590 nm using excitation at 530 nm.
Glucose-6-phosphate (G6P) quantification assay adapted from previous work (Zhu et al., 2009). Ten μl of G6P standards and extraction samples were pipetted to a 96-well plate, followed by the addition of 90 μl of a cocktail of 50 mM triethanolamine (pH 7.6), 1.0 mM MgCl2, 100 μM NADP+, 10 μM resazurin, 0.1 U/ml G6PD, and 0.2 U/ml diaphorase. The assays were incubated for 30 min, followed by measurement of fluorescence at 590 nm using excitation at 530 nm.
For all in vivo metabolic studies, age-matched WT and ΔE1HK1 littermates were used. For the glucose-tolerance test (GTT), mice were fasted for 16 hours and injected via an i.p. approach with a 20% dextrose (Millipore Sigma) solution in PBS at 2 g/kg body weight. For the insulin-tolerance test (ITT), mice were fasted for 4 hours and injected via an i.p. approach with a 0.1 U Humulin/ml PBS solution at 0.75 U/kg body weight (Lily).
Mitochondrial Isolation Kit for Tissue (Pierce) was used to purify mitochondrial and cytosolic protein subcellular fractions according to the Pierce manufacturer's protocol.
HK activity was determined, as previously described (Majewski et al., 2004). In brief, cells were plated on 12 cm dishes and allowed to attach overnight. The next day, cells were washed once with PBS and harvested by scraping and pelleted at 4000 rpm for 5 minutes. Cells were lysed by sonication, five pulses of 1 second, in 100 μl homogenization buffer: 0.2% Triton X-100, 0.5 mM EGTA, 10 mM D-(+)-glucose, 11.1 mM monothioglycerol, 45 mM Tris-HCl (pH 8.2), and 50 mM KH2PO4. After sonication, lysates were centrifuged at 8000 rpm for 5 minutes. HK activity was determined by the whole-cell lysate's ability to phosphorylate glucose over 2 minutes in an assay mixture with final concentrations of 50 mM triethanolamine chloride, 7.5 mM MgCl2, 0.5 mM EGTA, 11 mM monothioglycerol, 0.5 to 25 mM glucose, 6.6 mM ATP, 0.5 mg/mL NADP, and 0.5 U/mL G6PDH, pH 8.5. G6P formation was measured indirectly by NADPH production from G6PDH by measuring absorbance at 340 nm on a spectrophotometer and was normalized to protein concentration as determined by BCA protein assay kit (Fisher Scientific).
Data are presented as mean±SEM or SD as indicated. For a two-group comparison unpaired two-tailed Student's t-tests was used. For data with multiple groups (>2) or multiple treatments a one- or two-way ANOVA was used as indicated followed by Tukey's post-hoc test to determine p-values for individual comparisons. No statistical methods were used to predetermine sample size. All statistical analysis was performed using Graphpad Prism 8.0. p<0.05 was considered to be statistically significant and is presented as *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, or ns=not significant). Survival experiment was performed in Prism 8 using comparison of survival curves with Log-rank (Mantel-Cox) test. For in vivo experiments, animals were assigned to experimental groups using simple randomization, without investigator blinding. Hierarchical clustering and heatmaps for metabolomics data were generated using MetaboAnalyst 4.0 statistical software (Chong et al., 2019).
Aflalo, C., and Azoulay, H. (1998). Binding of Rat Brain Hexokinase to Recombinant Yeast Mitochondria: Effect of Environmental Factors and the Source of Porin. J. Bioenerg. Biomembr. 30, 245-255.
In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
Citations to a number of patent and non-patent references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.
The present application represents the national stage entry of PCT/US2021/071037, filed Jul. 28, 2021, which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/057,402, filed on Jul. 28, 2020. The contents of each are hereby incorporated by reference in their entireties.
This invention was made with government support under HL138982, HL127646, and HL132552 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/071037 | 7/28/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63057402 | Jul 2020 | US |
