Water channel structures analysed by electron crystallography

Background

The mechanisms underlying water transport through aquaporin (AQP) have been debated for two decades. The water permeation phenomenon of AQP seems inexplicable because the Grotthuss mechanism does not allow for simultaneous fast water permeability and inhibition of proton transfer through the hydrogen bonds of water molecules.

Scope of review

The AQP1 structure determined by electron crystallography provided the first insights into the proton exclusion mechanism despite fast water permeation. Although several studies have provided clues about the mechanism based on the AQP structure, each proposed mechanism remains incomplete. The present review is focused on AQP function and structure solved by electron crystallography in an attempt to fill the gaps between the findings in the absence and presence of lipids.

Major conclusions

Many AQP structures can be superimposed regardless of the determination method. The AQP fold is preserved even under conditions lacking lipids, but the water arrangement in the channel pore differs. The differences might be explained by dipole moments formed by the two short helices in the lipid bilayer. In addition, structure analyses of double-layered two-dimensional crystals of AQP suggest an array formation and cell adhesive function.

General significance

Electron crystallography findings not only have contributed to resolve some of the water permeation mechanisms, but have also elucidated the multiple functions of AQPs in the membrane. The roles of AQPs in the brain remain obscure, but their multiple activities might be important in the regulation of brain and other biological functions. This article is part of a Special Issue entitled Aquaporins.     

Intact and N- or C-terminal end truncated AQP0 function as open water channels and cell-to-cell adhesion proteins: End truncation could be a prelude for adjusting the refractive index of the lens to prevent spherical aberration

Background

Investigate the impact of natural N- or C-terminal post-translational truncations of lens mature fiber cell Aquaporin 0 (AQP0) on water permeability (Pw) and cell-to-cell adhesion (CTCA) functions.

Methods

The following deletions/truncations were created by site-directed mutagenesis (designations in parentheses): Amino acid residues (AA) 2–6 (AQP0-N-del-2-6), AA235–263 (AQP0-1-234), AA239–263 (AQP0-1-238), AA244–263 (AQP0-1-243), AA247–263 (AQP0-1-246), AA250–263 (AQP0-1-249) and AA260–263 (AQP0-1-259). Protein expression was studied using immunostaining, fluorescent tags and organelle-specific markers. Pw was tested by expressing the respective complementary ribonucleic acid (cRNA) in Xenopus oocytes and conducting osmotic swelling assay. CTCA was assessed by transfecting intact or mutant AQP0 into adhesion-deficient L-cells and performing cell aggregation and adhesion assays.

Results

AQP0-1-234 and AQP0-1-238 did not traffic to the plasma membrane. Trafficking of AQP0-N-del-2-6 and AQP0-1-243 was reduced causing decreased membrane Pw and CTCA. AQP0-1-246, AQP0-1-249 and AQP0-1-259 mutants trafficked properly and functioned normally. Pw and CTCA functions of the mutants were directly proportional to the respective amount of AQP0 expressed at the plasma membrane and remained comparable to those of intact AQP0 (AQP0-1-263).

Conclusions

Post-translational truncation of N- or C-terminal end amino acids does not alter the basal water permeability of AQP0 or its adhesive functions. AQP0 may play a role in adjusting the refractive index to prevent spherical aberration in the constantly growing lens.

General significance

Similar studies can be extended to other lens proteins which undergo post-translational truncations to find out how they assist the lens to maintain transparency and homeostasis for proper focusing of objects on to the retina.     

Aquaporin and brain diseases

Background

The presence of water channel proteins, aquaporins (AQPs), in the brain led to intense research in understanding the underlying roles of each of them under normal conditions and pathological conditions.

Scope of review

In this review, we summarize some of the recent knowledge on the 3 main AQPs (AQP1, AQP4 and AQP9), with a special focus on AQP4, the most abundant AQP in the central nervous system.

Major conclusions

AQP4 was most studied in several brain pathological conditions ranging from acute brain injuries (stroke, traumatic brain injury) to the chronic brain disease with autoimmune neurodegenerative diseases. To date, no specific therapeutic agents have been developed to either inhibit or enhance water flux through these channels. However, experimental results strongly underline the importance of this topic for future investigation. Early inhibition of water channels may have positive effects in prevention of edema formation in brain injuries but at later time points during the course of a disease, AQP is critical for clearance of water from the brain into blood vessels.

General significance

Thus, AQPs, and in particular AQP4, have important roles both in the formation and resolution of edema after brain injury. The dual, complex function of these water channel proteins makes them an excellent therapeutic target. This article is part of a Special Issue entitled Aquaporins.     

Deletion of aquaporin-4 in APP/PS1 mice exacerbates brain Aβ accumulation and memory deficits

Background

Preventing or reducing amyloid-beta (Aβ) accumulation in the brain is an important therapeutic strategy for Alzheimer’s disease (AD). Recent studies showed that the water channel aquaporin-4 (AQP4) mediates soluble Aβ clearance from the brain parenchyma along the paravascular pathway. However the direct evidence for roles of AQP4 in the pathophysiology of AD remains absent.

Results

Here, we reported that the deletion of AQP4 exacerbated cognitive deficits of 12-moth old APP/PS1 mice, with increases in Aβ accumulation, cerebral amyloid angiopathy and loss of synaptic protein and brain-derived neurotrophic factor in the hippocampus and cortex. Furthermore, AQP4 deficiency increased atrophy of astrocytes with significant decreases in interleukin-1 beta and nonsignficant decreases in interleukin-6 and tumor necrosis factor-alpha in hippocampal and cerebral samples.

Conclusions

These results suggest that AQP4 attenuates Aβ pathogenesis despite its potentially inflammatory side-effects, thus serving as a promising target for treating AD.

     

Human aquaporins: Regulators of transcellular water flow

Background

Emerging evidence supports the view that (AQP) aquaporin water channels are regulators of transcellular water flow. Consistent with their expression in most tissues, AQPs are associated with diverse physiological and pathophysiological processes.

Scope of review

AQP knockout studies suggest that the regulatory role of AQPs, rather than their action as passive channels, is their critical function. Transport through all AQPs occurs by a common passive mechanism, but their regulation and cellular distribution varies significantly depending on cell and tissue type; the role of AQPs in cell volume regulation (CVR) is particularly notable. This review examines the regulatory role of AQPs in transcellular water flow, especially in CVR. We focus on key systems of the human body, encompassing processes as diverse as urine concentration in the kidney to clearance of brain oedema.

Major conclusions

AQPs are crucial for the regulation of water homeostasis, providing selective pores for the rapid movement of water across diverse cell membranes and playing regulatory roles in CVR. Gating mechanisms have been proposed for human AQPs, but have only been reported for plant and microbial AQPs. Consequently, it is likely that the distribution and abundance of AQPs in a particular membrane is the determinant of membrane water permeability and a regulator of transcellular water flow.

General significance

Elucidating the mechanisms that regulate transcellular water flow will improve our understanding of the human body in health and disease. The central role of specific AQPs in regulating water homeostasis will provide routes to a range of novel therapies. This article is part of a Special Issue entitled Aquaporins.     

Unraveling aquaporin interaction partners

Background

Insight into protein–protein interactions (PPIs) is highly desirable in order to understand the physiology of cellular events. This understanding is one of the challenges in biochemistry and molecular biology today, especially for eukaryotic membrane proteins where hurdles of production, purification and structural determination must be passed.

Scope of review

We have explored the common strategies used to find medically relevant interaction partners of aquaporins (AQPs). The most frequently used methods to detect direct contact, yeast two-hybrid interaction assay and co-precipitation, are described together with interactions specifically found for the selected targets AQP0, AQP2, AQP4 and AQP5.

Major conclusions

The vast majority of interactions involve the aquaporin C-terminus and the characteristics of the interaction partners are strikingly diverse. While the well-established methods for PPIs are robust, a novel approach like bimolecular fluorescence complementation (BiFC) is attractive for screening many conditions as well as transient interactions. The ultimate goal is structural evaluation of protein complexes in order to get mechanistic insight into how proteins communicate at a molecular level.

General significance

What we learn from the human aquaporin field in terms of method development and communication between proteins can be of major use for any integral membrane protein of eukaryotic origin. This article is part of a Special Issue entitled Aquaporins.     

Renal aquaporins and water balance disorders

Background

Aquaporins (AQPs) are a family of proteins that can act as water channels. Regulation of AQPs is critical to osmoregulation and the maintenance of body water homeostasis. Eight AQPs are expressed in the kidney of which five have been shown to play a role in body water balance; AQP1, AQP2, AQP3, AQP4 and AQP7. AQP2 in particular is regulated by vasopressin.

Scope of review

This review summarizes our current knowledge of the underlying mechanisms of various water balance disorders and their treatment strategies.

Major conclusions

Dysfunctions of AQPs are involved in disorders associated with disturbed water homeostasis. Hyponatremia with increased AQP levels can be caused by diseases with low effective circulating blood volume, such as congestive heart failure, or osmoregulation disorders such as the syndrome of inappropriate secretion of antidiuretic hormone. Treatment consists of fluid restriction, demeclocycline and vasopressin type-2 receptor antagonists. Decreased AQP levels can lead to diabetes insipidus (DI), characterized by polyuria and polydipsia. In central DI, vasopressin production is impaired, while in gestational DI, levels of the vasopressin-degrading enzyme vasopressinase are abnormally increased. Treatment consists of the vasopressin analogue dDAVP. Nephrogenic DI is caused by the inability of the kidney to respond to vasopressin and can be congenital, but is most commonly acquired, usually due to lithium therapy. Treatment consists of sufficient fluid supply, low-solute diet and diuretics.

General significance

In recent years, our understanding of the underlying mechanisms of water balance disorders has increased enormously, which has opened up several possible new treatment strategies. This article is part of a Special Issue entitled Aquaporins.     

AQP1 Is Not Only a Water Channel: It Contributes to Cell Migration through Lin7/Beta-Catenin

Background

AQP1 belongs to aquaporins family, water-specific, membrane-channel proteins expressed in diverse tissues. Recent papers showed that during angiogenesis, AQP1 is expressed preferentially by microvessels, favoring angiogenesis via the increase of permeability In particular, in AQP1 null mice, endothelial cell migration is impaired without altering their proliferation or adhesion. Therefore, AQP1 has been proposed as a novel promoter of tumor angiogenesis.

Methods/Findings

Using targeted silencing of AQP1 gene expression, an impairment in the organization of F-actin and a reduced migration capacity was demonstrated in human endothelial and melanoma cell lines. Interestingly, we showed, for the first time, that AQP1 co-immunoprecipitated with Lin-7. Lin7-GFP experiments confirmed co-immunoprecipitation. In addition, the knock down of AQP1 decreased the level of expression of Lin-7 and β-catenin and the inhibition of proteasome contrasted partially such a decrease.

Conclusions/Significance

All together, our findings show that AQP1 plays a role inside the cells through Lin-7/β-catenin interaction. Such a role of AQP1 is the same in human melanoma and endothelial cells, suggesting that AQP1 plays a global physiological role. A model is presented.     

Macrophage elastase (MMP-12) in expanding murine adipose tissue

Background

Matrix metalloproteinases (MMPs) are known to play a role in adipose tissue development, but little information is available on the role of individual proteinases. Expansion of adipose tissue is associated with an increased macrophage content. Macrophage elastase (MMP-12) has an important role in macrophage infiltration, which induces pro-inflammatory effects in adipose tissue.

Methods

The role of MMP-12 was investigated in adipose tissues of MMP-12 deficient and wild-type control mice kept on normal chow or on high fat diet for 15 weeks.

Results

MMP-12 deficiency had no significant effect on total body weight or on subcutaneous (SC) or gonadal (GON) adipose tissue mass. Adipocyte and blood vessel size and density in SC and GON adipose tissues of obese mice were also comparable in MMP-12 deficient and control mice. Macrophage infiltration in SC and GON adipose tissues was not affected by MMP-12 deficiency, but the amount of crown-like structures (CLS) was significantly lower. MMP-12 deficiency did not affect elastin content in the extracellular matrix of SC or GON adipose tissue.

Conclusions

Adipose tissue mass and composition in mice with nutritionally induced obesity was not markedly affected by MMP-12 deficiency, except for an apparently lower degree of CLS.

General Significance

MMP-12 does not seem to be essential for macrophage infiltration in adipose tissue, but contributes to the formation of CLS surrounding moribund adipocytes.     

Different endocytic functions of AGEF-1 in C. elegans coelomocytes

Background

ADP-ribosylation factors (ARFs) are a family of small GTP-binding proteins that play roles in membrane dynamics and vesicle trafficking. AGEF-1, which is thought to act as a guanine nucleotide exchange factor of class I ARFs, is required for caveolin-1 body formation and receptor-mediated endocytosis in oocytes of Caenorhabditis elegans. This study explores additional roles of AGEF-1 in endocytic transport.

Methods

agef-1 expression was knocked down by using RNAi in C. elegans. Markers that allow analysis of endocytic transport in scavenger cells were investigated for studying the effect of AGEF-1 on different steps of membrane transport.

Results

Knockdown of AGEF-1 levels results in two apparent trafficking defects in coelomocytes of C. elegans. First, there is a delay in the uptake of solutes from the extracellular medium. Second, there is a dramatic enlargement of the sizes of lysosomes, even though lysosomal acidification is normal and degradation still occurs.

Conclusion

Our results suggest that AGEF-1 regulates endosome/lysosome fusion or fission events, in addition to earlier steps in endocytic transport.

General significance

AGEF-1 is the first identified GTPase regulator that functions at the lysosome fusion or fission stage of the endocytic pathway. Our study provides insight into lysosome dynamics in C. elegans.     

Site-specifically C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death

Background

Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging.

Methods

AnxA5 and the control protein were recombinantly expressed with a C-terminal “Sel-tag”, the tetrapeptide –Gly-Cys-Sec-Gly–COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with ¹¹C for dynamic in vivo PET studies.

Results

AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The ¹¹C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease.

Conclusions

These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy.

General significance

The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of ¹¹C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.     

Aquaporins in cancer

Background

The aquaporins (AQPs) are a family of 13 small hydrophobic integral transmembrane water channel proteins involved in transcellular and transepithelial water movement, transport of fluid and cell migration.

Scope of the review

This review article summarizes our knowledge concerning the involvement of AQPs in tumor growth, angiogenesis and metastatic process.

Major conclusions

Tumor cells types express AQPs and a positive correlation exists between histological tumor grade and the AQP expression. Moreover, AQPs are involved also in tumor edema formation and angiogenesis in several solid and hematological tumors.

General significance

AQPs inhibition in endothelial and tumor cells might limit tumor growth and spread, suggesting a potential therapeutic use in the treatment of tumors. This article is part of a Special Issue entitled Aquaporins.     

LOX-1 deletion and macrophage trafficking in atherosclerosis

Background

Atherosclerosis is associated with macrophage accumulation. LOX-1 has been shown to induce macrophage attachment, and its deletion (LOX-1 knockout, KO) reduces atherosclerosis in LDLr KO mice fed a high cholesterol diet. We examined differences in macrophage trafficking in age-matched wild type, LOX-1 KO, LDLr KO, and LDLr/LOX-1 double KO mice.

Methods

Sections of aortas of mice fed high cholesterol diet were collected at weeks 0, 4, 8, 12 and 19 and analyzed by immunohistochemistry and flow cytometry.

Results

In the LDLr KO mice aorta, CD68 positivity (macrophage accumulation) increased over time up to 12 weeks, and then the accumulation fell modestly but significantly. The periaortal fat and adventitia showed more CD68 positivity than the media and intima. This pattern was also evident in the non-atherosclerotic areas. Importantly, LOX-1 KO and LDLr–LOX-1 double KO mice showed diminished CD68 positivity in comparison to wild type and LDLR KO mice, respectively. Further, macrophages from LOX-1 KO mice revealed a marked reduction in migration (vs. macrophages from wild type mice) in in vitro migration assay.

Conclusions

LOX-1 deletion translates into reduction in macrophage trafficking in the aorta of LDLr KO mice. Most of the macrophage trafficking appears in the subadventitial regions.     

Melatonin renders neuroprotection by protein kinase C mediated aquaporin-4 inhibition in animal model of focal cerebral ischemia

Aim

Aquaporin-4(AQP4) expression in the brain with relation to edema formation following focal cerebral ischemia was investigated. Studies have shown that brain edema is one of the significant factors in worsening stroke outcomes. While many mechanisms may aggravate brain injury, one such potential system may involve AQP4 up regulation in stroke patients that could result in increased edema formation. Post administration of melatonin following ischemic stroke reduces AQP4 mediated brain edema and confers neuroprotection.

Materials and methods

An in-silico approach was undertaken to confirm effective melatonin-AQP4 binding. Rats were treated with 5 mg/kg, i.p. melatonin or placebo at 30 min prior, 60 min post and 120 min post 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Rats were evaluated for battery of neurological and motor function tests just before sacrifice. Brains were harvested for infarct size estimation, water content measurement, biochemical analysis, apoptosis study and western blot experiments.

Key findings

Melatonin at 60 min post ischemia rendered neuroprotection as evident by reduction in cerebral infarct volume, improvement in motor and neurological deficit and reduction in brain edema. Furthermore, ischemia induced surge in levels of nitrite and malondialdehyde (MDA) were also found to be significantly reduced in ischemic brain regions in treated animals. Melatonin potentiated intrinsic antioxidant status, inhibited acid mediated rise in intracellular calcium levels, decreased apoptotic cell death and also markedly inhibited protein kinase C (PKC) influenced AQP4 expression in the cerebral cortex and dorsal striatum.

Significance

Melatonin confers neuroprotection by protein kinase C mediated AQP4 inhibition in ischemic stroke.     

Serum ferritin: Past,present and future

Background

Serum ferritin was discovered in the 1930s, and was developed as a clinical test in the 1970s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases.

Scope of review

In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool.

Major conclusions

Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer.

General significance

Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.     

DCs sensitized with mPD-L1-Ig fusion protein improve the effect of heart transplantation in mice by promoting the generation of T-reg cells

Purpose

To detect the effects of DCs sensitized by mPD-L1-Ig fusion protein in heart transplantation in mice as well as its mechanisms.

Method

The mPD-L1-IgG1 construct was used to build a yeast expression system, and the fusion protein was expressed by secretion after the transfection of the GS115 yeast strain, purified by affinity chromatography and ion exchange chromatography, and assayed by SDS–PAGE and Western blot. The ability of the fusion protein to bind to the acceptor PD-1 was tested by ELISA, and the ability of the fusion protein to inhibit the function of T cells was tested by mixed lymphocyte reaction (MLR).

Results

We used the new PD-L1-IgG1 fusion protein to sensitize imDCs and maintained the immature state of DCs, so as to induce stable and effective immune tolerance to heart transplantation. After the treatment of DCs by mPD-L1-Ig in vitro, the levels of CD80, CD40 and I-Ab expression on DCs are relatively weaker, the ability of DCs to stimulates the proliferation of allogeneic spleen T cells was significantly decreased (P < 0.01), and the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) secreted by induced allogeneic T cells were significantly decreased (P < 0.01). An in vivo experiment also revealed that DCs sensitized by mPD-L1-IgG1 could prolong the survival time of a transplanted heart to 17.8 ± 1.12 days, and alleviate the pathological change of the cardiac allografts compared with other three groups.

Conclusion

DCs sensitized by the yeast-expressed mPD-L1-Ig fusion protein are shown to alleviate the cardiac allograft rejection in mice.     

Intracellular Ca2+ transients in delta-sarcoglycan knockout mouse skeletal muscle

Background

δ-Sarcoglycan (δ-SG) knockout (KO) mice develop skeletal muscle histopathological alterations similar to those in humans with limb muscular dystrophy. Membrane fragility and increased Ca²⁺ permeability have been linked to muscle degeneration. However, little is known about the mechanisms by which genetic defects lead to disease.

Methods

Isolated skeletal muscle fibers of wild-type and δ-SG KO mice were used to investigate whether the absence of δ-SG alters the increase in intracellular Ca²⁺ during single twitches and tetani or during repeated stimulation. Immunolabeling, electrical field stimulation and Ca²⁺ transient recording techniques with fluorescent indicators were used.

Results

Ca²⁺ transients during single twitches and tetani generated by muscle fibers of δ-SG KO mice are similar to those of wild-type mice, but their amplitude is greatly decreased during protracted stimulation in KO compared to wild-type fibers. This impairment is independent of extracellular Ca²⁺ and is mimicked in wild-type fibers by blocking store-operated calcium channels with 2-aminoethoxydiphenyl borate (2-APB). Also, immunolabeling indicates the localization of a δ-SG isoform in the sarcoplasmic reticulum of the isolated skeletal muscle fibers of wild-type animals, which may be related to the functional differences between wild-type and KO muscles.

Conclusions

δ-SG has a role in calcium homeostasis in skeletal muscle fibers.

General significance

These results support a possible role of δ-SG on calcium homeostasis. The alterations caused by the absence of δ-SG may be related to the pathogenesis of muscular dystrophy.     

Increased Expression of AQP 1 and AQP 5 in Rat Lungs Ventilated with Low Tidal Volume is Time Dependent

Background and Goals

Mechanical ventilation (MV) can induce or worsen pulmonary oedema. Aquaporins (AQPs) facilitate the selective and rapid bi-directional movement of water. Their role in the development and resolution of pulmonary oedema is controversial. Our objectives are to determine if prolonged MV causes lung oedema and changes in the expression of AQP 1 and AQP 5 in rats.

Methods

25 male Wistar rats were subjected to MV with a tidal volume of 10 ml/kg, during 2 hours (n = 12) and 4 hours (n = 13). Degree of oedema was compared with a group of non-ventilated rats (n = 5). The expression of AQP 1 and AQP 5 were determined by western immunoblotting, measuring the amount of mRNA (previously amplified by RT-PCR) and immunohistochemical staining of AQPs 1 and 5 in lung samples from all groups.

Results

Lung oedema and alveolar-capillary membrane permeability did not change during MV. AQP-5 steady state levels in the western blot were increased (p<0.01) at 2 h and 4 h of MV. But in AQP-1 expression these differences were not found. However, the amount of mRNA for AQP-1 was increased at 2 h and 4 h of MV; and for AQP 5 at 4 h of MV. These findings were corroborated by representative immunohistochemical lung samples.

Conclusion

In lungs from rats ventilated with a low tidal volume the expression of AQP 5 increases gradually with MV duration, but does not cause pulmonary oedema or changes in lung permeability. AQPs may have a protective effect against the oedema induced by MV.     

Characterization of a gene encoding prohibitin in silkworm, Bombyx mori

Background

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown.

Methods

To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry.

Results

BmPHB gene has an ORF of 825 bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva's silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth.

Conclusions

The expression of BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm.

General significance

Studies on prohibitin have been still restricted to a few specific insects and insect cell lines such as Drosophila, Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, B. mori.     

Methionine oxidation and reduction in proteins

Background

Cysteine and methionine are the two sulfur containing amino acids in proteins. While the roles of protein-bound cysteinyl residues as endogenous antioxidants are well appreciated, those of methionine remain largely unexplored.

Scope

We summarize the key roles of methionine residues in proteins.

Major conclusion

Recent studies establish that cysteine and methionine have remarkably similar functions.

General significance

Both cysteine and methionine serve as important cellular antioxidants, stabilize the structure of proteins, and can act as regulatory switches through reversible oxidation and reduction. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.