LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function. M itochondria descend from ancient Gram-negative bacteria that, through endosymbiosis, became permanent residents of eukaryotic cells (1-3). As a consequence, mitochondria and Gram-negative bacteria share several biochemical features, including DNA organization, core metabolism, and a doublemembrane architecture. In Gram-negative bacteria, but not in mitochondria, a mesh-like layer of peptidoglycan is deposited between the outer and inner membrane, offering protection against mechanical stress. Following endosymbiosis, the peptidoglycan layer lost its structural importance, and was subsequently eliminated from the early eukaryotic cell. Although eukaryotes lack peptidoglycan, proteins deriving from the penicillin-binding protein (PBP) family (4) are found in all major eukaryotic lineages, including vertebrates (5).The bacterial PBPs constitute a large family of serine proteases that is distinguished by 3 conserved amino acid motifs that contribute to the formation of the catalytic site. The -SXXKmotif contains the catalytic serine residue, which undergoes reversible acylation through substrate binding, whereas the -[SY]X[NT]-and the - [KH][ST]G-motifs contribute to substrate docking (4). PBPs catalyze the hydrolysis or transpeptidation of the terminal D-alanyl-D-alanine moiety in peptidoglycan stem peptides. The acceptor for the transpeptidation reaction is the -amino group of lysine or diaminopimelate in an adjacent stem peptide. Through these reactions, PBPs contribute to modulate the extent of peptidoglycan cross-linking during bacterial cell division and cell wall elongation (4).In contrast, the function of PBP homologues in eukaryotic organisms remains largely unexplored. Amino acid sequence analyses show that the 3 conserved amino acid motifs required for catalytic activity are conserved in all eukaryotic PBP homologues (5), suggesting that they can function as active-site serine enzymes. Within the metazoan division, nematodes harbor the largest number of PBP homologues (5), and in Caenorhabditis elegans the PBP homologue LACT-1 may be involved in pathogen recognition (6). LACTB is the only PBP homologue of mammals, and LACTB has been identified in all mammalian genomes sequenced to date (5,7,8). Recently, a causative link between LACTB and obesity was detected through gene coexp...

Background: Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins.

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SUMMARY:Progression to hormone-refractory growth of prostate cancer has been suggested to be mediated by androgen receptor (AR) gene alterations. We analyzed AR for mutations and amplifications in 21 locally recurrent prostate carcinomas treated with orchiectomy, estrogens, or a combination of orchiectomy and estramustine phosphate using fluorescence in situ hybridization, single-strand conformation polymorphism, and DNA sequence analyses. Amplification was observed in 4 of 16 (25%) and amino acid changing mutations was observed in 7 of 21 (33%) of the tumors, respectively. Two (50%) tumors with AR amplification also had missense mutation of the gene. Four of five (80%) cancers that were treated with a combination of orchiectomy and estramustine phosphate had a mutation clustered at codons 514 to 533 in the N-terminal domain of AR. In functional studies, these mutations did not render AR more sensitive to testosterone, dihydrotestosterone, androstenedione, or ␤-estradiol. Tumors treated by orchiectomy had mutations predominantly in the ligand-binding domain. In summary, we found molecular alterations of AR in more than half of the prostate carcinomas that recurred locally. Some tumors developed both aberrations, possibly enhancing the cancer cell to respond efficiently to low levels of androgens. Furthermore, localization of point mutations in AR seems to be influenced by the type of treatment. (Lab Invest 2002, 82:1591-1598. P rostate cancer (CaP) is the most common male malignancy in the Western world, and its prognosis greatly depends on at which stage the disease is diagnosed. Even in the era of prostate specific antigen (PSA) screening, advanced disease is diagnosed in 20 to 40% of patients with CaP when cure by radical prostatectomy or radiotherapy is not considered possible anymore. In addition, recurrence rates of CaP after radical surgery or radiotherapy approach 25 to 50% (Dennis and Griffiths, 2000;Määttänen et al, 1999;Scardino et al, 1994). For these patients, androgen deprivation therapy (ADT) remains the only effective palliative treatment. ADT is generally achieved by either surgical or chemical castration, but for patients with widespread, metastasized disease, the cytotoxic drug estramustine phosphate (EMP) is occasionally combined with ADT (Kuhn et al, 1994;Murphy et al, 1986). Although all ADTs are initially effective, in most patients, CaP progresses within months or a few years (Dennis and Griffiths, 2000;Scardino et al, 1994). Molecular mechanisms of ADT failure are not comprehensively known. Previous studies have suggested a link between androgen receptor (AR) gene and ADT failure (Culig et al, 1993;Elo et al, 1995;Koivisto et al, 1997;Schoenberg et al, 1994;Suzuki et al, 1993Suzuki et al, , 1996Taplin et al, 1995Taplin et al, , 1999Visakorpi et al, 1995;Wallen et al, 1999), and two main mechanisms by which CaP cells could adapt and sensitize AR signaling pathway for growth in low levels of androgens have been proposed. First, AR gene amplification has been shown to lead to increased expr...

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.

We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.

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Small carboxyl-terminal domain (CTD) phosphatase 2 (SCP2) was identified and verified as a protein that interacts with the androgen receptor (AR). Ectopic expression of SCP2 or two other family members, SCP1 and SCP3, attenuated AR transcriptional activity in LNCaP cells and were recruited in an androgen-and AR-dependent fashion onto the prostate-specific antigen (PSA) promoter. Silencing SCP2 and SCP1 by short hairpin RNAs increased androgen-dependent transcription of the PSA gene and augmented AR loading onto the PSA promoter and enhancer. SCP2 also attenuated glucocorticoid receptor (GR) function, and its silencing increased dexamethasonemediated PSA mRNA accumulation and GR loading onto the PSA enhancer in LNCaP 1F5 cells. SCP2 silencing was accompanied by augmented recruitment and earlier cycling of RNA polymerase II on the promoter. Ser 5 phosphorylation of the RNA polymerase II CTD, a process necessary for initiation of transcription elongation, occurred significantly earlier in SCP2-silenced than parental LNCaP cells. Collectively, our results suggest that SCP2 is involved in promoter clearance during steroid-activated transcription.