Samie R. Jaffrey

Protein S -nitrosylation: a physiological signal for neuronal nitric oxide

Nitric oxide (NO) has been linked to numerous physiological and pathophysiological events that are not readily explained by the well established effects of NO on soluble guanylyl cyclase. Exogenous NO S-nitrosylates cysteine residues in proteins, but whether this is an important function of endogenous NO is unclear. Here, using a new proteomic approach, we identify a population of proteins that are endogenously S-nitrosylated, and demonstrate the loss of this modification in mice harbouring a genomic deletion of neuronal NO synthase (nNOS). Targets of NO include metabolic, structural and signalling proteins that may be effectors for neuronally generated NO. These findings establish protein S-nitrosylation as a physiological signalling mechanism for nNOS.

The Biotin Switch Method for the Detection of S-Nitrosylated Proteins

Many of the effects of nitric oxide are mediated by the direct modification of cysteine residues resulting in an adduct called a nitrosothiol. Here, we describe a novel method for detecting proteins that contain nitrosothiols. In this three-step procedure, nitrosylated cysteines are converted to biotinylated cysteines. Biotinylated proteins can then be detected by immunoblotting or can be purified by avidin-affinity chromatography. We include examples of the detection of S-nitrosylated proteins in brain lysates after in vitro S-nitrosylation, as well as the detection of endogenous S-nitrosothiols in selected neuronal proteins.

Haem oxygenase-1 prevents cell death by regulating cellular iron

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.

PIN: An Associated Protein Inhibitor of Neuronal Nitric Oxide Synthase

The neurotransmitter functions of nitric oxide are dependent on dynamic regulation of its biosynthetic enzyme, neuronal nitric oxide synthase (nNOS). By means of a yeast two-hybrid screen, a 10-kilodalton protein was identified that physically interacts with and inhibits the activity of nNOS. This inhibitor, designated PIN, appears to be one of the most conserved proteins in nature, showing 92 percent amino acid identity with the nematode and rat homologs. Binding of PIN destabilizes the nNOS dimer, a conformation necessary for activity. These results suggest that PIN may regulate numerous biological processes through its effects on nitric oxide synthase activity.

Local translation of RhoA regulates growth cone collapse.

Neuronal development requires highly coordinated regulation of the cytoskeleton within the developing axon. This dynamic regulation manifests itself in axonal branching, turning and pathfinding, presynaptic differentiation, and growth cone collapse and extension. Semaphorin 3A (Sema3A), a secreted guidance cue that primarily functions to repel axons from inappropriate targets, induces cytoskeletal rearrangements that result in growth cone collapse. These effects require intra-axonal messenger RNA translation. Here we show that transcripts for RhoA, a small guanosine triphosphatase (GTPase) that regulates the actin cytoskeleton, are localized to developing axons and growth cones, and this localization is mediated by an axonal targeting element located in the RhoA 3′ untranslated region (UTR). Sema3A induces intra-axonal translation of RhoA mRNA, and this local translation of RhoA is necessary and sufficient for Sema3A-mediated growth cone collapse. These studies indicate that local RhoA translation regulates the neuronal cytoskeleton and identify a new mechanism for the regulation of RhoA signalling.

CAPON: a protein associated with neuronal nitric oxide synthase that regulates its interactions with PSD95.

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ-PDZ domain interaction between PSD95 and nNOS. Here, we report the identification of a novel nNOS-associated protein, CAPON, which is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes.

Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling

Protein ubiquitination is a post-translational modification (PTM) that regulates various aspects of protein function by different mechanisms. Characterization of ubiquitination has lagged behind that of smaller PTMs, such as phosphorylation, largely because of the difficulty of isolating and identifying peptides derived from the ubiquitinated portion of proteins. To address this issue, we generated a monoclonal antibody that enriches for peptides containing lysine residues modified by diglycine, an adduct left at sites of ubiquitination after trypsin digestion. We use mass spectrometry to identify 374 diglycine-modified lysines on 236 ubiquitinated proteins from HEK293 cells, including 80 proteins containing multiple sites of ubiquitination. Seventy-two percent of these proteins and 92% of the ubiquitination sites do not appear to have been reported previously. Ubiquitin remnant profiling of the multi-ubiquitinated proteins proliferating cell nuclear antigen (PCNA) and tubulin α-1A reveals differential regulation of ubiquitination at specific sites by microtubule inhibitors, demonstrating the effectiveness of our method to characterize the dynamics of lysine ubiquitination.

Dexras1: A G protein specifically coupled to neuronal nitric oxide synthase via CAPON

Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins. Here we identify a selective interaction of the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. We find that Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). We also find that nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced.

Dynamic m6A mRNA methylation directs translational control of heat shock response

The most abundant mRNA post-transcriptional modification is N6-methyladenosine (m6A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m6A along mRNAs results in relatively less methylation in the 5′ untranslated region (5′UTR) compared to other regions. However, whether and how 5′UTR methylation is regulated is poorly understood. Despite the crucial role of the 5′UTR in translation initiation, very little is known about whether m6A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5′UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5′UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m6A ‘reader’. Upon heat shock stress, the nuclear YTHDF2 preserves 5′UTR methylation of stress-induced transcripts by limiting the m6A ‘eraser’ FTO from demethylation. Remarkably, the increased 5′UTR methylation in the form of m6A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m6A modification site in the 5′UTR enables translation initiation independent of the 5′ end N7-methylguanosine cap. The elucidation of the dynamic features of 5′UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m6A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

Fluorescence imaging of cellular metabolites with RNA

Cellular metabolites are detected within living cells by fluorescent RNA-based ligand-binding sensors. Genetically encoded sensors are powerful tools for imaging intracellular metabolites and signaling molecules. However, developing sensors is challenging because they require proteins that undergo conformational changes upon binding the desired target molecule. We describe an approach for generating fluorescent sensors based on Spinach, an RNA sequence that binds and activates the fluorescence of a small-molecule fluorophore. We show that these sensors can detect a variety of different small molecules in vitro and in living cells. These RNAs constitute a versatile approach for fluorescence imaging of small molecules and have the potential to detect essentially any cellular biomolecule.