Molecular insights into substrate translocation in an elevator-type metal transporter.

The Zrt/Irt-like protein (ZIP) metal transporters are key players in maintaining the homeostasis of a panel of essential microelements. The prototypical ZIP from Bordetella bronchiseptica (BbZIP) is an elevator transporter, but how the metal substrate moves along the transport pathway and how the transporter changes conformation to allow alternating access remain to be elucidated. Here, we combine structural, biochemical, and computational approaches to investigate the process of metal substrate translocation along with the global structural rearrangement.

Molecular insights into substrate translocation in an elevator-type metal transporter.

The Zrt/Irt-like protein (ZIP) metal transporters are key players in maintaining the homeostasis of a panel of essential microelements. The prototypical ZIP from (BbZIP) is an elevator transporter, but how the metal substrate moves along the transport pathway and how the transporter changes conformation to allow alternating access remain to be elucidated. Here, we combined structural, biochemical, and computational approaches to investigate the process of metal substrate translocation along with the global structural rearrangement.

Irreversible inactivation of lactate racemase by sodium borohydride reveals reactivity of the nickel-pincer nucleotide cofactor.

The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from (LarA) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarA, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH) irreversibly inactivates LarA accompanied by a rapid color change of the enzyme.

Expression, purification, crystallization of a ZIP metal transporter from Bordetella bronchiseptica (BbZIP).

Structural studies of the ZIPs have greatly improved the understanding of the working mechanism for this functionally important metal transporter family. In this chapter, we describe the procedures to overexpress, purify, and crystallize a representative bacterial ZIP from Bordetella bronchiseptica (BbZIP), the structure of which was the first one that revealed the common structural framework of the transmembrane domain conserved within the entire ZIP family. We also discuss the considerations when we designed these experiments and compare the approaches used in this study with those commonly used in other works.

Cell-based transport assay to study kinetics and substrate specificity of human ZIPs.

Kinetic study of human ZIPs is crucial for understanding the transport mechanism and the molecular basis of substrate specificity. In this chapter, we describe the detailed experimental procedures for functional studies of two human ZIPs, including the zinc-preferring ZIP4 and the multi-metal transporter ZIP8, by using the cell-based transport assays. Kinetic study of ZIP4 is elaborated in the first section; in the second section, comparison of ZIP4 and ZIP8 in terms of the zinc/cadmium selectivity is performed by using an internal competition assay adapted from the established cell-based approach.

Rational engineering of an elevator-type metal transporter ZIP8 reveals a conditional selectivity filter critically involved in determining substrate specificity.

Engineering of transporters to alter substrate specificity as desired holds great potential for applications, including metabolic engineering. However, the lack of knowledge on molecular mechanisms of substrate specificity hinders designing effective strategies for transporter engineering. Here, we applied an integrated approach to rationally alter the substrate preference of ZIP8, a Zrt-/Irt-like protein (ZIP) metal transporter with multiple natural substrates, and uncovered the determinants of substrate specificity.

High-resolution structure of a mercury cross-linked ZIP metal transporter reveals delicate motions and metal relay for regulated zinc transport.

Zrt-/Irt-like protein (ZIP) divalent metal transporters play a central role in maintaining trace element homeostasis. The prototypical ZIP from (BbZIP) is an elevator-type transporter, but the dynamic motions and detailed transport mechanism remain to be elucidated. Here, we report a high-resolution crystal structure of a mercury-crosslinked BbZIP variant at 1.

Structural insights into the elevator-type transport mechanism of a bacterial ZIP metal transporter.

The Zrt-/Irt-like protein (ZIP) family consists of ubiquitously expressed divalent metal transporters critically involved in maintaining systemic and cellular homeostasis of zinc, iron, and manganese. Here, we present a study on a prokaryotic ZIP from Bordetella bronchiseptica (BbZIP) by combining structural biology, evolutionary covariance, computational modeling, and a variety of biochemical assays to tackle the issue of the transport mechanism which has not been established for the ZIP family. The apo state structure in an inward-facing conformation revealed a disassembled transport site, altered inter-helical interactions, and importantly, a rigid body movement of a 4-transmembrane helix (TM) bundle relative to the other TMs.

Rational Design and Synthesis of D-galactosyl Lysophospholipids as Selective Substrates and non-ATP-competitive Inhibitors of Phosphatidylinositol Phosphate Kinases.

Phosphatidylinositol phosphate kinases (PIPKs) produce lipid signaling molecules and have been attracting increasing attention as drug targets for cancer, neurodegenerative diseases, and viral infection. Given the potential cross-inhibition of kinases and other ATP-utilizing enzymes by ATP-competitive inhibitors, targeting the unique lipid substrate binding site represents a superior strategy for PIPK inhibition. Here, by taking advantage of the nearly identical stereochemistry between myo-inositol and D-galactose, we designed and synthesized a panel of D-galactosyl lysophospholipids, one of which was found to be a selective substrate of phosphatidylinositol 4-phosphate 5-kinase.

Zinc transporter mutations linked to acrodermatitis enteropathica disrupt function and cause mistrafficking.

ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, acrodermatitis enteropathica (AE). These mutations occur not only in the conserved transmembrane zinc transport machinery, but also in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs.

Hyperphosphorylated tau aggregation and cytotoxicity modulators screen identified prescription drugs linked to Alzheimer's disease and cognitive functions.

The neurodegenerative Alzheimer's disease (AD) affects more than 30 million people worldwide. There is thus far no cure or prevention for AD. Aggregation of hyperphosphorylated tau in the brain correlates with the cognitive decline of patients of AD and other neurodegenerative tauopathies.

Molecular Basis of Zinc-Dependent Endocytosis of Human ZIP4 Transceptor.

Nutrient transporters can be rapidly removed from the cell surface via substrate-stimulated endocytosis as a way to control nutrient influx, but the molecular underpinnings are not well understood. In this work, we focus on zinc-dependent endocytosis of human ZIP4 (hZIP4), a zinc transporter that is essential for dietary zinc uptake. Structure-guided mutagenesis and internalization assay reveal that hZIP4 per se acts as the exclusive zinc sensor, with the transport site's being responsible for zinc sensing.

Asymmetric functions of a binuclear metal center within the transport pathway of a human zinc transporter ZIP4.

Metal clusters are exploited by numerous metalloenzymes for catalysis, but it is not common to utilize a metal cluster for substrate transport across membrane. The recent crystal structure of a prototypic Zrt-/Irt-like protein (ZIP) metal transporter from Bordetella bronchiseptica (BbZIP) revealed an unprecedented binuclear metal center (BMC) within the transport pathway. Here, through a combination of bioinformatics, biochemical and structural approaches, we concluded that the two physically associated metal-binding sites in the BMC of human ZIP4 (hZIP4) zinc transporter exert different functions: one conserved transition metal-binding site acts as the transport site essential for activity, whereas the variable metal-binding site is required for hZIP4's optimal activity presumably by serving as a secondary transport site and modulating the properties of the primary transport site.

The histidine-rich loop in the extracellular domain of ZIP4 binds zinc and plays a role in zinc transport.

The Zrt-/Irt-like protein (ZIP) family mediates zinc influx from extracellular space or intracellular vesicles/organelles, playing a central role in systemic and cellular zinc homeostasis. Out of the 14 family members encoded in human genome, ZIP4 is exclusively responsible for zinc uptake from dietary food and dysfunctional mutations of ZIP4 cause a life-threatening genetic disorder, Acrodermatitis Enteropathica (AE). About half of the missense AE-causing mutations occur within the large N-terminal extracellular domain (ECD), and our previous study has shown that ZIP4-ECD is crucial for optimal zinc uptake but the underlying mechanism has not been clarified.

Structural insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense mutation of PIP5Kγ.

Signaling molecule phosphatidylinositol 4,5-bisphosphate is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3, caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5Kγ. So far, the exact role of this aspartate residue has yet to be elucidated.

Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway.

Zrt/Irt-like proteins (ZIPs) play fundamental roles in metal metabolism/homeostasis and are broadly involved in numerous physiological and pathological processes. The lack of high-resolution structure of the ZIPs hinders understanding of the metal transport mechanism. We report two crystal structures of a prokaryotic ZIP in lipidic cubic phase with bound metal substrates (Cd at 2.

The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K's activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K.

Structural insights of ZIP4 extracellular domain critical for optimal zinc transport.

The ZIP zinc transporter family is responsible for zinc uptake from the extracellular milieu or intracellular vesicles. The LIV-1 subfamily, containing nine out of the 14 human ZIP proteins, is featured with a large extracellular domain (ECD). The critical role of the ECD is manifested by disease-causing mutations on ZIP4, a representative LIV-1 protein.

In vitro aggregation assays using hyperphosphorylated tau protein.

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks.

Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex.

Phosphopeptide enrichment with TiO2-modified membranes and investigation of tau protein phosphorylation.

Selective enrichment of phosphopeptides prior to their analysis by mass spectrometry (MS) is vital for identifying protein phosphorylation sites involved in cellular regulation. This study describes modification of porous nylon substrates with TiO2 nanoparticles to create membranes that rapidly enrich phosphopeptides. Membranes with a 22-mm diameter bind 540 nmol of phosphoangiotensin and recover 70% of the phosphopeptides in mixtures with a 15-fold excess of nonphosphorylated proteins.

Intramembrane proteolytic cleavage of a membrane-tethered transcription factor by a metalloprotease depends on ATP.

Regulated intramembrane proteolysis (RIP) involves cleavage of a transmembrane segment of a protein. RIP governs diverse processes in a wide variety of organisms and is carried out by different types of intramembrane proteases (IPs), including a large family of metalloproteases. The Bacillus subtilis SpoIVFB protein is a putative metalloprotease that cleaves membrane-tethered Pro-sigma(K), releasing sigma(K) to direct transcription of genes necessary for spore formation.

Salt intake augments hypotensive effects of transient receptor potential vanilloid 4: functional significance and implication.

To test the hypothesis that activation of the transient receptor potential vanilloid 4 (TRPV4) channel conveys a hypotensive effect that is enhanced during salt load, male Wistar rats fed a normal-sodium (0.5%) or high-sodium (HS; 4%) diet for 3 weeks were given 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), a specific TRPV4 activator, in the presence or absence of capsazepine, a selective TRPV1 blocker, ruthenium red, a TRPV4 blocker, or TRPV4 small hairpin RNA that selectively knockdowns TRPV4. 4 alpha-PDD (1, 2.