Polycarboxylates enhance beetle antifreeze protein activity

Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N',N',N',N'-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn(2+) coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed.     

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of α-intercalated cells of the kidney collecting duct leads to the defect of the Cl⁻/ exchange and the failure of proton (H⁺) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney α-intercalated cells.     

Homology modeling of Mycoplasma pneumoniae enolase and its molecular interaction with human plasminogen

Alpha (α)-enolase (e), a glycolytic enzyme, has an alternative role as a surface receptor of several bacteria mediating plasminogen (pg) binding. It is also recognized as a virulence factor of some pathogenic bacteria facilitating plasminogen activation and host cell invasion. A mycoplasmal α-enolase is also a plasminogen binding protein. Molecular interactions of enolase from Mycoplasma pneumoniae with host plasminogen would be useful for exploring the pathogen-host interaction. In an attempt to identify plasminogen binding sites of M. pneumoniae enolase, homology modeling and docking studies were conducted to obtain modeled structures of the M. pneumoniae enolase-plasminogen complex. The refined model was validated further by standard methods. Molecular docking revealed hydrogen bonding of eLys70-pgTyr50, eAsn165-pgThr66, eAla168-pgGlu21, eAsp17-pgLys70, and eAsn213-pgPro68/pgAsn69. Substantial decreases in accessible surface area (ASA) were observed and in concurrence with hydrogen bond pattern. These findings provide a detailed prediction of key residues that interact at the protein-protein interface. Our theoretical prediction is consistent with known biochemical data. The predicted interaction complex can be of great assistance in understanding structural insights, which is necessary to pathogen and host-component interaction. The ability of M. pneumoniae enolase to bind plasminogen may be indicative of an important role in invasion of this pathogen to host.     

Interaction of reduced nicotinamide adenine dinucleotide with an antifreeze protein from Dendroides canadensis: mechanistic implication of antifreeze activity enhancement

Antifreeze proteins (AFPs) found in many organisms can noncolligatively lower the freezing point of water without altering the melting point. The difference between the depressed freezing point and the melting point, termed thermal hysteresis (TH), is usually a measure of the antifreeze activity of AFPs. Certain low molecular mass molecules and proteins can further enhance the antifreeze activity of AFPs. Interaction between an enhancer and arginine is known to play an important role in enhancing the antifreeze activity of an AFP from the beetle Dendroides canadensis (DAFP-1). Here, we examined the enhancement effects of several prevalent phosphate-containing coenzymes on the antifreeze activity of DAFP-1. β-Nicotinamide adenine dinucleotide (reduced) (NADH) is identified as the most efficient enhancer of DAFP-1, which increases the antifreeze activity of DAFP-1 by around 10 times. Examination of the enhancement abilities of a series of NADH analogs and various molecular fragments of NADH reveals that the modifications of nicotinamide generate a series of highly efficient enhancers, though none as effective as NADH itself, and the whole molecular structure of NADH is necessary for its highly efficient enhancement effect. We also demonstrated a 1:1 binding between DAFP-1 and NADH. The binding was characterized by high-performance liquid chromatography (HPLC) using the gel filtration method of Hummel and Dreyer. The data analysis suggests binding between DAFP-1 and NADH with a dissociation constant in the micromolar range. Interactions between DAFP-1 and NADH are discussed along with molecular mechanisms of enhancer action.     

Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 (kAE1) to the Cell Surface

Kidney anion exchanger 1 (kAE1) plays an important role in acid–base homeostasis by mediating chloride/bicarbornate (Cl^?/HCO₃^?) exchange at the basolateral membrane of α‐intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease – distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans‐Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral‐related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non‐polarized kidney cells. By using RNA interference, co‐immunoprecipitation, yellow fluorescent protein‐based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP‐1 mu1A, AP‐3 mu1, AP‐4 mu1 and clathrin (but not AP‐1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral‐related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP‐1 mu1A, AP‐3 mu1, AP‐4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid‐secreting α‐intercalated cells.