TuKiet T. Lam

Identification of Novel Interactions in HIV-1 Capsid Protein Assembly by High-resolution Mass Spectrometry

The pleomorphic nature of the immature and mature HIV-1 virions has made it difficult to characterize intersubunit interactions using traditional approaches. While the structures of isolated domains are known, the challenge is to identify intersubunit interactions and thereby pack these domains into supramolecular structures. Using high-resolution mass spectrometry, we have measured the amide hydrogen exchange protection factors for the soluble capsid protein (CA) and CA assembled in vitro. Comparison of the protection factors as well as chemical crosslinking experiments has led to a map of the subunit/subunit interfaces in the assembled tubes. This analysis provides direct biochemical evidence for the homotypic N domain and C domain interactions proposed from cryo-electron microscopy image reconstruction of CA tubes. Most significantly, we have identified a previously unrecognized intersubunit N domain-C domain interaction. The detection of this interaction reconciles previously discrepant biophysical and genetic data.

Key interactions in HIV-1 maturation identified by hydrogen-deuterium exchange

To characterize the intersubunit interactions underlying assembly and maturation in HIV-1, we determined the amide hydrogen exchange protection pattern of capsid protein in the immature virion and the mature virion using mass spectrometry. Alterations in protection upon maturation provide evidence for the maturation-induced formation of an interaction between the N- and C-terminal domains in half of the capsid molecules, indicating that only half of the capsid protein is assembled into the conical core.

A Gut Lipid Messenger Links Excess Dietary Fat to Dopamine Deficiency

Food as Reward Why does ice cream taste so good? High-fat foods activate a reward circuit in the brain involving dopamine, a neurotransmitter that regulates pleasure. Overconsumption of high-fat foods is thought to dampen this dopamine-induced reward sensation, leading to compensatory consumption of even more high-fat foods. The mechanisms by which dietary fat in the gut “talks” to the dopamine reward circuit are unclear. Tellez et al. (p. 800) suggest that an intestinal lipid messenger called oleoylethanolamine (OEA) may play a role—at least in mice. Mice on a high-fat diet had unusually low levels of intestinal OEA and exhibited deficient dopaminergic responses to gut stimulation with high-fat lipids. Infusion of OEA into these mice restored the dopaminergic response, and mice that had been accustomed to a high-fat diet began to eat more low-fat foods. In mice, a high-fat diet functionally disrupts a gut lipid that controls the brain’s perception of the reward value of food. Excessive intake of dietary fats leads to diminished brain dopaminergic function. It has been proposed that dopamine deficiency exacerbates obesity by provoking compensatory overfeeding as one way to restore reward sensitivity. However, the physiological mechanisms linking prolonged high-fat intake to dopamine deficiency remain elusive. We show that administering oleoylethanolamine, a gastrointestinal lipid messenger whose synthesis is suppressed after prolonged high-fat exposure, is sufficient to restore gut-stimulated dopamine release in high-fat–fed mice. Administering oleoylethanolamine to high-fat–fed mice also eliminated motivation deficits during flavorless intragastric feeding and increased oral intake of low-fat emulsions. Our findings suggest that high-fat–induced gastrointestinal dysfunctions play a key role in dopamine deficiency and that restoring gut-generated lipid signaling may increase the reward value of less palatable, yet healthier, foods.

Diversification of a Salmonella Virulence Protein Function by Ubiquitin-Dependent Differential Localization

Many bacterial pathogens and symbionts utilize type III secretion systems to deliver bacterial effector proteins into host cells. These effector proteins have the capacity to modulate a large variety of cellular functions in a highly regulated manner. Here, we report that the phosphoinositide phosphatase SopB, a Salmonella Typhimurium type III secreted effector protein, diversifies its function by localizing to different cellular compartments in a ubiquitin-dependent manner. We show that SopB utilizes the same enzymatic activity to modulate actin-mediated bacterial internalization and Akt activation at the plasma membrane and vesicular trafficking and intracellular bacterial replication at the phagosome. Thus, by exploiting the host cellular machinery, Salmonella Typhimurium has evolved the capacity to broaden the functional repertoire of a virulence factor to maximize its ability to modulate cellular functions.

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

This study identifies an unusual sulfur-based chemical as a novel and specific inhibitor of the tyrosine phosphatase STEP and shows that it can improve the cognitive function of a mouse model of Alzheimers disease.

Functional visualization of viral molecular motor by hydrogen-deuterium exchange reveals transient states

Molecular motors undergo cyclical conformational changes and convert chemical energy into mechanical work. The conformational dynamics of a viral packaging motor, the hexameric helicase P4 of dsRNA bacteriophage φ8, was visualized by hydrogen-deuterium exchange and high-resolution mass spectrometry. Concerted changes of exchange kinetics revealed a cooperative unit that dynamically links ATP-binding sites and the central RNA-binding channel. The cooperative unit is compatible with a structure-based model in which translocation is mediated by a swiveling helix. Deuterium labeling also revealed the transition state associated with RNA loading, which proceeds via opening of the hexameric ring. The loading mechanism is similar to that of other hexameric helicases. Hydrogen-deuterium exchange provides an important link between time-resolved spectroscopic observations and high-resolution structural snapshots of molecular machines.

Mapping of protein: protein contact surfaces by hydrogen/deuterium exchange, followed by on-line high-performance liquid chromatography-electrospray ionization fourier-transform ion-cyclotron-resonance mass analysis

For protein complexes too large, uncrystallizable/insoluble, or low concentration for conventional X-ray diffraction or nuclear magnetic resonance analysis, the contact surface(s) may be mapped by comparing H/2H exchange rate (and thus solvent accessibility) of backbone amide hydrogens in free vs. complexed protein(s). The protein is first exposed to 2H2O, allowed to exchange for each of several reaction periods, and then digested with pepsin. The extent and rate of H/2H exchange is determined by measuring the increase in mass with H/2H exchange period for each of the peptides. Here, we present an experimental protocol that combines rapid (to minimize back-exchange) HPLC front-end separation with ultrahigh-resolution mass analysis (needed to distinguish the isotopic distributions of dozens of peptides simultaneously). The method is used to study the assembled human immunodeficiency virus type capsid protein (CA) and its soluble form.

Endothelial cell palmitoylproteomic identifies novel lipid-modified targets and potential substrates for protein acyl transferases.

Rationale: Protein S-palmitoylation is the posttranslational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well-understood, partly because of technological limits on palmitoylprotein detection. Objective: To develop a method using acyl-biotinyl exchange technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in ECs. Methods and Results: More than 150 putative palmitoyl proteins were identified in ECs using acyl-biotinyl exchange and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase-1, an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine-6 prevents palmitoylation, leads to reduction in superoxide dismutase-1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for superoxide dismutase-1 palmitoylation. Moreover, we used acyl-biotinyl exchange to search for substrates of particular protein acyl transferases in ECs. We found that palmitoylation of the cell adhesion protein platelet endothelial cell adhesion molecule-1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of platelet endothelial cell adhesion molecule-1 at the cell surface. Conclusions: Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important posttranslational lipid modification in EC biology.

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn60. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn70/Asn104 flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn60 reduces adhesion, N-glycans at Asn70/Asn104 of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

Significance Aldosterone produces distinct adaptive responses in volume depletion and hyperkalemia. Mutations in with-no-lysine (WNK) kinases or ubiquitin ligases containing Cullin 3 (CUL3) and Kelch-like 3 (KLHL3) cause a Mendelian disease featuring hypertension and hyperkalemia due to constitutive renal salt reabsorption and inhibited K+ secretion. WNKs modulate activities of aldosterone-regulated electrolyte flux pathways, and WNK levels are regulated by CUL3/KLHL3; disease-causing mutations prevent WNK degradation. This manuscript shows that angiotensin II (AII), a hormone produced only in volume depletion, induces PKC-mediated phosphorylation of KLHL3, preventing WNK degradation and phenocopying KLHL3 mutations. These findings provide a mechanism by which AII signaling alters WNK4, promoting increased renal salt reabsorption and reduced K+ secretion. Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K+ reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K+ secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)—components of an E3 ubiquitin ligase complex that targets WNKs for degradation—cause constitutively increased renal salt reabsorption and impaired K+ secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3S433 phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K+ secretion in the setting of volume depletion.