Research Progress in the Structure and Functions of Prohibitin

Journal of Reproduction & Contraception

http://www.RandC.cn

2010 Jun.; 21(2):117-124

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Research Progress in the Structure and Functions of Prohibitin Jia-xian OU, Si-fei HUANG, Hong CHEN Department of Anatomy & Histo-Embryology, Shanghai Medical School, Fudan University, Shanghai 200032, China

Prohibitin is named due to the negative regulatory role of its gene products in cell proliferation. Prohibitin gene is located at q21 of chromosome 17 in human beings and the protein is found at mitochondria, nucleus and cytoplasm. Due to its size and ring-shaped structure, prohibitin protein defines functional subcompartments in mitochondria. Its subunits, PHB1 and PHB2, suppress cell proliferation as in the protein itself. Nevertheless, recent investigation suggests that prohibitin protein enhances cell proliferation as well. It has also been found to suppress cell apoptosis by reducing cytochrome C release via the avoidance of mitochondrial crista remodeling which is facilitated through type 1 optic atrophy protein (OPA1). Acting as a binding site for ubiquitin, prohibitin protein regulates protein degradation by proteasome. Examples are the degradations of sperm mitochondria in a fertilized ovum or those of an abnormal sperm. Key words: prohibitin; cell proliferation; mitochondrial cristae; type 1 optic atrophy protein (OPA1); cell apoptosis ‘Prohibitin’ is so called due to the negative regulatory role of its gene product in cell proliferation. In human beings, the prohibitin gene has been located at q21 of chromosome 17[1]. Meanwhile, its protein has been found at the mitochondria, nucleus and cytoplasm[2]. Structurally speaking, the prohibitin gene gives rise to two transcripts which differ in lengths. Such variation arises due to a difference in the length of their 3' UTRs. In actively proliferating This project is supported by NSFC grant J0730860 Corresponding author: Hong CHEN; E-mail: [email protected] 117

tissues and cells, a higher level of the longer transcript can be detected. This implies that 3' UTR may function as a trans-acting riboregulator in cell cycle[3]. As a protein, prohibitin consists of PHB1 and PHB2. It has been found ubiquitously expressed in eukaryotic cells[4,5]. Currently, prohibitin protein is seen related to cell proliferation and mitochondrial functions. In this essay, advances in the research of prohibitin will be elaborated.

The structure and assembly of prohibitin Prohibitin gene occurs as two alleles. One is cleaved at a polymorphic intronic EcoRI site and exhibits an exon 6-associated SSCP. The other is not cleaved at the EcoRI site and has a different exon 6 SSCP pattern[6]. Prohibitin gene is evolutionary conserved. The comparison between prohibitin genes in rats and humans discovers eight highly conserved regions with four of them corresponding to binding sites of known transcriptional control proteins (CCAAT box, ‘SV40’ sites and two Spl sites). The exon-intron boundaries of both prohibitin genes are found highly conserved with splice junctions identically positioned. Their promoters are also found deficient of a TATA box[7]. Prohibitin mRNA codes for the synthesis of prohibitin protein which contains 272 amino acid residues. The primary structure of prohibitin protein lacks motifs typical for signal sequences, nuclear localization signals, ATP-binding sites or transcription factors[8]. It consists of two homologous proteins namely prohibitin 1 (PHB1) and prohibitin 2 (PHB2) which share over 50% identical amino acid sequences (Figure 1A). When one prohibitin gene is deleted, both prohibitin subunits are lost. This implies the interdependence of the existence of both subunits. Structurally speaking, each subunit has a hydrophobic stretch at the amino terminal end that anchors the protein to the inner membrane of the mitochondria and a large carboxy terminal domain that exposes to the intermembrane space. The carboxyl terminal constitutes a PHB domain/stomatin prohibitin flotillin-HflC/K (SPFH) domain and a coiled-coil region, the latter structure being essential for the assembly of prohibitin complexes in yeast. When PHB1 is first imported into yeast mitochondria, it associates with Tim8/13 complexes in the intermembrane space which function as molecular chaperones in the biogenesis of inner and outer membrane proteins. Then, PHB1 is inserted into the inner membrane through the mediation by TIM23 translocase. PHB2 subunits assemble with PHB1 to form dimmers of about 120 000 that act as building blocks of prohibitin complexes. Such complexes occur in rings of alternating subunit composition and each of them has an average diameter of around 2025 nm (Figure 1B, C).

The functions of PHB1, PHB2 and prohibitin protein PHB1 and PHB2 are assembling parts of prohibitin protein. Therefore, it is essential to 118

understand the roles of PHB1 and PHB2 before unraveling the physiological roles of prohibitin protein. The functions of PHB1 and PHB2 Regulation of cell cycle PHB1 mRNA level decreases in rat liver cells upon entry into cell cycle after partial hepatectomy[9]. At the same time, microinjection of PHB1 mRNA suppresses the progression of cell cycle[10]. Moreover, human PHB1 has been seen in a yeast two-hybrid screen as a retinoblastoma-binding protein that inhibits E2F transcriptional activation[11]. Related studies also state how PHB1 protein binds with E2F and Raf-1 kinase directly to suppress the progression of cell cycle[11]. All these lead to a conclusion stating that PHB1 is a negative regulator of the cell cycle. Currently some scholars relate the antiproliferative role of PHB1 to the 3' UTR of its mRNA but not to the protein coding region[12,13]. Such is supported by the functional inhibition assays on truncated wild-type mRNA transcripts, 3' UTR specific wild-type mRNA transcripts and mutated transcripts of PHB1[12]. An experiment which involves the microinjection of the protein coding region of PHB1 also verifies the assumption above. PHB2, on the other hand, has been isolated from a yeast two-hybrid screen. It is seen as an estrogen receptor binding protein which represses transcriptional activation[14]. Participation in receptor mediated cell signaling In studies using co-immunoprecipitation, both PHB1 protein and PHB2 protein have 119

been associated with cell-surface IgM instead of cell-surface IgD on murine B lymphocytes. This implies, to a certain extent, the difference in biological events elicited after the engagement of either of these receptors[15]. The functions of prohibitin protein complex Regulation of cell cycle and apoptosis Suppression of cell proliferation As stated above, PHB1 and PHB2 suppress cell proliferation. Thus, it is commonly accepted that prohibitin protein complex shares the same role. In fact, the isolation of prohibitin cDNA from murine liver cells has been based on such a characteristic [9]. The locus which associates with prohibitin’s anti-proliferative role is 17q21. In most sporadic breast cancer patients whose age of onset is below 36, the loss of heterozygosity at the long arm of chromosome 17 can be found[1]. Also, gene mutations like deletions which occur at this locus in many cases of breast cancer suggests the possibility of the gene being a tumor suppressor[12]. Enhancement of cell proliferation[16] Such enhancement was originally proposed due to mouse embryonic fibroblast (MEF) proliferation suppression upon the knockout of PHB2 gene and the subsequent loss of PHB1 and PHB2 subunits. Initially, it was associated with non-mitochondrial prohibitin. However, when identical result was observed in MEFs whose mitochondria lacked PHB1 and PHB2, the association between cell proliferation enhancement and mitochondrial prohibitin was established. Suppression of cell apoptosis Cytochrome C is released from mitochondria at the early stage of cell apoptosis. This is enhanced via the remodeling of mitochondrial cristae. MEFs lacking prohibitin have mitochondria that release more cytochrome C due to the remodeling of mitochondrial cristae. This facilitates apoptosis and so prohibitin is seen as an anti-apoptotic protein[16]. Influence over morphology of mitochondrial cristae The morphology of mitochondrial cristae is associated with some conservative proteins at the inner and outer mitochondrial membranes such as mitofusin and OPA1. These conservative proteins contribute in the formation and remodeling of the reticulated mitochondrial network[17]. While OPA1 works by enhancing mitochondrial fusion[18], its functions rely on the dynamic equilibrium between L-OPA1 and S-OPA1[19-21]. Prohibitin protein prevents the hydrolysis of L-OPA1 into S-OPA1. This stabilizes OPA1 and thus enhances the influence of OPA1 over the morphology of mitochondrial cristae[17]. At a lack or decrease of prohibitin protein, abnormality in the morphology of mitochondrial cristae can arise. One example is the accumulation of fragmented mitochondria in HeLa cells and MEFs 120

lacking prohibitin protein[16,22]. Another example is the presence of abnormal, vesicular mitochondrial cristae in the body wall muscle cells of C. elegans upon decreased prohibitin expression[23]. Influence over respiratory chain In MEFs and yeasts, prohibitin is not essential for the maintenance of mitochondrial membrane potential and respiratory activity[16,24,25]. However, recent research of prohibitin on angiogenesis displays its importance in the maintenance of mitochondrial membrane potential and complex I activity in endothelial cells: upon the knock-down of PHB1, the above indices decrease yet the production of reactive oxygen species (ROS) increases[26]. The influence of prohibitin over the respiratory chain may be exhibited through OPA1 since a Drosophila model for optic atrophy caused by mutations in Opa1 has been found with an increase in ROS production and mitochondrial fragmentation[27]. Influence over proteolysis in mitochondrial inner membrane ATP-dependent m-AAA protease is structurally conservative with its catalytic domains present at the mitochondrial inner membrane. It mainly controls the protein quality at the mitochondrial inner membrane and it is vital in the biogenesis of mitochondria. Prohibitin protein complex binds with m-AAA protease[28-30] (Figure 2) and if deleted in yeast, serves to increase the rate of proteolysis[28]; implying that prohibitin plays a regulatory role in the degradation of membrane proteins. Furthermore, as m-AAA protease is associated with the processing of OPA1 [16] and a decrease in prohibitin expression reduces L-OPA1, an association between prohibitin protein, L-OPA1 and m-AAA protease may be established.

In contrast to prohibitins, m-AAA protease subunits expose their catalytic domains to the matrix space. The binding of the m-AAA protease to the inner or outer surface of ringshaped prohibitin complexes remains to be established[2] Figure 2 Supercomplex of prohibitins with the ATP-dependent m-AAA protease

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Definition of functional subcompartments The size and ring-shaped structure of prohibitin complexes suggest they may act as scaffolds which define functional subcompartments. This is important for specific processes in the inner membrane and outer membrane of the mitochondria[25,31]. For example, the genetic interaction of prohibitin with assembly factors like ATP10 and ATP23 (which code for substrate-specific chaperones in the assembly of F1F0-ATP synthase) prevents the hazardous effects that arise from F0-assembly intermediates[31]. Regulation of sperm metabolism[32] Prohibitin protein acts as a binding site of ubiquitin. As ubiquinated protein is degraded through proteasome, this suggests a relationship between prohibitin and protein degradation. After fertilization, ubiquitin that is attached to the prohibitin localized at the inner membrane of sperm mitochondria is exposed. This then leads to mitochondrial degradation, suggesting that prohibitin is related to the maternal inheritance of mtDNA. Aside from that, damage at the disulphide linkage in the mitochondria of abnormal sperm leads to the early exposure of prohibitin in the epididymis. This brings about their early destruction. However, due to the presence of stable disulphide linkage in the mitochondria of normal sperm, prohibitin is left unexposed. This prevents their early destruction. Such phenomena relate prohibitin to sperm quality control. The content above summarizes the functions of prohibitin protein complex, PHB1 and PHB2. In brief, these functions emphasize the influence of prohibitin over mitochondria, implying that the stability of mitochondrial morphology and mitochondrial functions serves to be an important factor which governs the roles of prohibitin. Back in the beginning, the discovery and nomenclature of prohibitin have arisen from its antiproliferative function. However, further studies bring us closer to understand prohibitin: the relationship between prohibitin and OPA1 affects the morphology and functions of mitochondria, suggesting that a close association exists between prohibitin, OPA1 and mitochondria. Moreover, prohibitin expression decreases in ageing, implying an intimate relationship between prohibitin, mitochondria and ageing[33]. Indeed, deeper investigation of prohibitin will widen our understanding regarding its roles and physiological importance. At the same time, the relationship between prohibitin and mitochondria can be further elaborated.

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