Visualizing mitochondrial heme flow through GAPDH in living cells and its regulation by NO.

Iron protoporphyrin IX (heme) is a redox-active cofactor that is bound in mammalian cells by GAPDH and allocated by a process influenced by physiologic levels of NO. This impacts the activity of many heme proteins including indoleamine dioxygenase-1 (IDO1), a redox enzyme involved in immune response and tumor growth. To gain further understanding we created a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after labeling could indicate its heme binding by fluorescence quenching.

Visualizing Mitochondrial Heme Flow through GAPDH to Targets in Living Cells and its Regulation by NO.

Iron protoporphyrin IX (heme) is an essential cofactor that is chaperoned in mammalian cells by GAPDH in a process regulated by NO. To gain further understanding we generated a tetra-Cys human GAPDH reporter construct (TC-hGAPDH) which after being expressed and labeled with fluorescent FlAsH reagent could indicate heme binding by fluorescence quenching. When purified or expressed in HEK293T mammalian cells, FlAsH-labeled TC-hGAPDH displayed physical, catalytic, and heme binding properties like native GAPDH and its heme binding (2 mol per tetramer) quenched its fluorescence by 45-65%.

Nitric oxide and heme-NO stimulate superoxide production by NADPH oxidase 5.

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g.

Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones.

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear.

GAPDH delivers heme to soluble guanylyl cyclase.

Soluble guanylyl cyclase (sGC) is a key component of NO-cGMP signaling in mammals. Although heme must bind in the sGC β1 subunit (sGCβ) for sGC to function, how heme is delivered to sGCβ remains unknown. Given that GAPDH displays properties of a heme chaperone for inducible NO synthase, here we investigated whether heme delivery to apo-sGCβ involves GAPDH.

Heat shock protein 90 regulates soluble guanylyl cyclase maturation by a dual mechanism.

The enzyme soluble guanylyl cyclase (sGC) is a heterodimer composed of an α subunit and a heme-containing β subunit. It participates in signaling by generating cGMP in response to nitric oxide (NO). Heme insertion into the β1 subunit of sGC (sGCβ) is critical for function, and heat shock protein 90 (HSP90) associates with heme-free sGCβ (apo-sGCβ) to drive its heme insertion.

Functional Studies on Steroid Receptors.

Nuclear receptors play an important role in prostate cancer and the androgen receptor is a key transcription factor in regulation of cellular events. Androgen receptor-associated coregulators may be upregulated or downregulated in prostate cancer. Altered expression of regulators may potentiate androgen-induced proliferation, migration, and invasion.

A comprehensive analysis of coregulator recruitment, androgen receptor function and gene expression in prostate cancer.

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood.

Intratumoral and Intertumoral Genomic Heterogeneity of Multifocal Localized Prostate Cancer Impacts Molecular Classifications and Genomic Prognosticators.

Background: Next-generation sequencing is revealing genomic heterogeneity in localized prostate cancer (CaP). Incomplete sampling of CaP multiclonality has limited the implications for molecular subtyping, stratification, and systemic treatment.

Objective: To determine the impact of genomic and transcriptomic diversity within and among intraprostatic CaP foci on CaP molecular taxonomy, predictors of progression, and actionable therapeutic targets.

Regulators of Androgen Action Resource: a one-stop shop for the comprehensive study of androgen receptor action.

Androgen receptor (AR) is a ligand-activated transcription factor that is the main target for treatment of non-organ-confined prostate cancer (CaP). Failure of life-prolonging AR-targeting androgen deprivation therapy is due to flexibility in steroidogenic pathways that control intracrine androgen levels and variability in the AR transcriptional output. Androgen biosynthesis enzymes, androgen transporters and AR-associated coregulators are attractive novel CaP treatment targets.

A bipartite interaction between Hsp70 and CHIP regulates ubiquitination of chaperoned client proteins.

The ubiquitin ligase CHIP plays an important role in cytosolic protein quality control by ubiquitinating proteins chaperoned by Hsp70/Hsc70 and Hsp90, thereby targeting such substrate proteins for degradation. We present a 2.91 Å resolution structure of the tetratricopeptide repeat (TPR) domain of CHIP in complex with the α-helical lid subdomain and unstructured tail of Hsc70.

Crystal structure of the nucleotide-binding domain of mortalin, the mitochondrial Hsp70 chaperone.

Mortalin, a member of the Hsp70-family of molecular chaperones, functions in a variety of processes including mitochondrial protein import and quality control, Fe-S cluster protein biogenesis, mitochondrial homeostasis, and regulation of p53. Mortalin is implicated in regulation of apoptosis, cell stress response, neurodegeneration, and cancer and is a target of the antitumor compound MKT-077. Like other Hsp70-family members, Mortalin consists of a nucleotide-binding domain (NBD) and a substrate-binding domain.

Inhibition of Mcl-1 promotes senescence in cancer cells: implications for preventing tumor growth and chemotherapy resistance.

Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS).

Diet enriched with omega-3 fatty acids alleviates convulsion symptoms in epilepsy patients.

Purpose: We examined whether a dietary supplement containing omega-3 polyunsaturated fatty acids (n-3 PUFAs) can alleviate and/or reduce the frequency of epileptic seizures in patients with central nervous system (CNS) diseases treated with anticonvulsive drugs (ACDs).

Methods: A special spread containing 65% n-3 PUFAs was added to the daily diet. The patients consumed 5 g of this spread at every breakfast for 6 months.