The Role of the Transport System in the Control of the Source–Sink Relations in Pinus sylvestris

Morphophysiological correlations were studied in medium-aged (20- to 60-year-old) Scots pine trees under the northern taiga conditions. Under various ecological conditions, pine trees developed a well-balanced structure, with close linear relationships between needle and root weight and their cross-section areas in all components of the continuous transport network (the coefficient of determination was between 0.88 and 0.999). When the annual cycle of soluble and insoluble carbohydrate contents was followed in various pine tissues, the total concentrations of soluble and insoluble carbohydrates were maintained at constant and tissue-specific levels, except in the growth period. The maximum level of carbohydrates was observed in all tissues at the beginning of rapid growth, and the minimum, at growth cessation. The qualitative composition and amount of carbohydrates matched the phenological phases of development and were not affected by the ecological growth conditions pertinent to the particular environment. The authors conclude that assimilate synthesis and partitioning are related to structural development, and the state of sink centers determines the attracting capacity, whereas the transport network, from roots to needles, and its conducting capacity are essential for the realization of systemic relationships and the control over growth and development in Pinus sylvestris L.     

Polymorphism of Y-Chromosomal Diallelic Loci in Populations of the Volga–Ural Region

Three diallelic polymorphisms of human Y chromosome (DYS287(Y Alupolymorphism, YAP), T/C transition at the RBF5locus (Tat), and G/A transition at the LLY22locus) were studied in eight ethnic populations of the Volga–Ural region, representing Turkic branch of the Altai (Bashkirs, Tatars, and Chuvashes) and Finno-Ugric branch of the Uralic linguistic family (Maris, Mordovians, Udmurts, Komi-Zyryans, and Komi-Permyaks), and in the group of Slavic migrants, belonging to the Indo-European linguistic family (Russians). The Volga–Ural populations were characterized by a low frequency of the Y chromosome Aluinsertion. Examination of an association between the Alupolymorphism and Tatmutation revealed absolute YAP–/C linkage. Analysis of the haplotype frequency distribution patterns constructed from the data on the DYS287and RBF5polymorphisms revealed substantial differences between Udmurts and other populations. The differences were also observed between Komi-Zyryans and the populations of Bashkirs, Mordovians, Komi-Permyaks, and Russians. Analysis of the degree of genetic differentiation pointed to high level of genetic differentiation of the male lineages of the Finno–Ugric ethnic groups. The data on the linkage between the RBF5locus and the LLY22locus mutations indicated the common origin of the Tatmutationin Bashkirs, Mordovians, Udmurts, and Komi-Zyryans, and of a number of ancestralCallele-bearing Y chromosomes in Tatars, Maris, and Chuvashes.     

Polymorphism of the Y-Chromosome Diallelic Loci in Ethnic Groups of the Altai–Sayan Region

Using the data on five diallellic Y-chromosome loci (DYS199,92R7, SRY1532, RBF5, and DYS287) polymorphism, genetic structures of the five Turkic-speaking ethnic groups of the Altai–Sayan upland (Tuvinians, Sojots, Shorians, Khakassians, and Southern Altaians (Altai-Kizhi), were described. The gene pools of the populations examined were characterized by the presence of pronounced paleo-Caucasoid component (92R7-T-lineages). The frequency of this component increased westward, reaching more than 70% in Shorians and Southern Altaians. Haplotype TAT-C (RBF5 locus) was observed in all populations, except Shorians, with the frequencies varying from 5.4% in Altai-Kizhi to 18.8% in Khakassians. The Alu-insertion in the DYS287 locus was revealed only in the Altaian sample with the frequency of 3.3%. It was established that the Altai–Sayan populations studied split into two statistically significantly different groups. One of the groups was represented by Tuvinians, Sojots, and Khakassians, while another one was comprised of Shorians and Altaians.     

Synergistic and Independent Actions of Multiple Terminal Nucleotidyl Transferases in the 3’ Tailing of Small RNAs in Arabidopsis

All types of small RNAs in plants, piwi-interacting RNAs (piRNAs) in animals and a subset of siRNAs in Drosophila and C. elegans are subject to HEN1 mediated 3’ terminal 2’-O-methylation. This modification plays a pivotal role in protecting small RNAs from 3’ uridylation, trimming and degradation. In Arabidopsis, HESO1 is a major enzyme that uridylates small RNAs to trigger their degradation. However, U-tail is still present in null hen1 heso1 mutants, suggesting the existence of (an) enzymatic activities redundant with HESO1. Here, we report that UTP: RNA uridylyltransferase (URT1) is a functional paralog of HESO1. URT1 interacts with AGO1 and plays a predominant role in miRNA uridylation when HESO1 is absent. Uridylation of miRNA is globally abolished in a hen1 heso1 urt1 triple mutant, accompanied by an extensive increase of 3’-to-5’ trimming. In contrast, disruption of URT1 appears not to affect the heterochromatic siRNA uridylation. This indicates the involvement of additional nucleotidyl transferases in the siRNA pathway. Analysis of miRNA tailings in the hen1 heso1 urt1 triple mutant also reveals the existence of previously unknown enzymatic activities that can add non-uridine nucleotides. Importantly, we show HESO1 may also act redundantly with URT1 in miRNA uridylation when HEN1 is fully competent. Taken together, our data not only reveal a synergistic action of HESO1 and URT1 in the 3’ uridylation of miRNAs, but also independent activities of multiple terminal nucleotidyl transferases in the 3’ tailing of small RNAs and an antagonistic relationship between uridylation and trimming. Our results may provide further insight into the mechanisms of small RNA 3’ end modification and stability control.     

Contacts in Death: The Role of the ER–Mitochondria Axis in Acetic Acid-Induced Apoptosis in Yeast

Endoplasmic reticulum–mitochondria contact sites have been a subject of increasing scientific interest since the discovery that these structures are disrupted in several pathologies. Due to the emerging data that correlate endoplasmic reticulum–mitochondria contact sites function with known events of the apoptotic program, we aimed to dissect this interplay using our well-established model of acetic acid-induced apoptosis in Saccharomyces cerevisiae. Until recently, the only known tethering complex between ER and mitochondria in this organism was the ER–mitochondria encounter structure (ERMES). Following our results from a screening designed to identify genes whose deletion rendered cells with an altered sensitivity to acetic acid, we hypothesized that the ERMES complex could be involved in cell death mediated by this stressor. Herein we demonstrate that single ablation of the ERMES components Mdm10p, Mdm12p and Mdm34p increases the resistance of S. cerevisiae to acetic acid-induced apoptosis, which is associated with a prominent delay in the appearance of several apoptotic markers. Moreover, abrogation of Mdm10p or Mdm34p abolished cytochrome c release from mitochondria. Since these two proteins are embedded in the mitochondrial outer membrane, we propose that the ERMES complex plays a part in cytochrome c release, a key event of the apoptotic cascade. In all, these findings will aid in targeted therapies for diseases where apoptosis is disrupted, as well as assist in the development of acetic acid-resistant strains for industrial processes.     

The Role of Gap Junctions in Endothelial–Stromal Cell Interactions

Human Physiology - Gap junctions are one of the most highly specialized intercellular communications providing not only electrical coupling but also metabolic cooperation between cells due to the...     

Requirements of the cytosolic iron–sulfur cluster assembly pathway in Arabidopsis

The assembly of iron–sulfur (Fe–S) clusters requires dedicated protein factors inside the living cell. Striking similarities between prokaryotic and eukaryotic assembly proteins suggest that plant cells inherited two different pathways through endosymbiosis: the ISC pathway in mitochondria and the SUF pathway in plastids. Fe–S proteins are also found in the cytosol and nucleus, but little is known about how they are assembled in plant cells. Here, we show that neither plastid assembly proteins nor the cytosolic cysteine desulfurase ABA3 are required for the activity of cytosolic aconitase, which depends on a [4Fe–4S] cluster. In contrast, cytosolic aconitase activity depended on the mitochondrial cysteine desulfurase NFS1 and the mitochondrial transporter ATM3. In addition, we were able to complement a yeast mutant in the cytosolic Fe–S cluster assembly pathway, dre2, with the Arabidopsis homologue AtDRE2, but only when expressed together with the diflavin reductase AtTAH18. Spectroscopic characterization showed that purified AtDRE2 could bind up to two Fe–S clusters. Purified AtTAH18 bound one flavin per molecule and was able to accept electrons from NAD(P)H. These results suggest that the proteins involved in cytosolic Fe–S cluster assembly are highly conserved, and that dependence on the mitochondria arose before the second endosymbiosis event leading to plastids.     

The Role of Aromatic–Aromatic Interactions in Strand–Strand Stabilization of β-Sheets

Aromatic–aromatic interactions have long been believed to play key roles in protein structure, folding, and binding functions. However, we still lack full understanding of the contributions of aromatic–aromatic interactions to protein stability and the timing of their formation during folding. Here, using an aromatic ladder in the β-barrel protein, cellular retinoic acid-binding protein 1 (CRABP1), as a case study, we find that aromatic π stacking plays a greater role in the Phe65–Phe71 cross-strand pair, while in another pair, Phe50–Phe65, hydrophobic interactions are dominant. The Phe65–Phe71 pair spans β-strands 4 and 5 in the β-barrel, which lack interstrand hydrogen bonding, and we speculate that it compensates energetically for the absence of strand–strand backbone interactions. Using perturbation analysis, we find that both aromatic–aromatic pairs form after the transition state for folding of CRABP1, thus playing a role in the final stabilization of the β-sheet rather than in its nucleation as had been earlier proposed. The aromatic interaction between strands 4 and 5 in CRABP1 is highly conserved in the intracellular lipid-binding protein (iLBP) family, and several lines of evidence combine to support a model wherein it acts to maintain barrel structure while allowing the dynamic opening that is necessary for ligand entry. Lastly, we carried out a bioinformatics analysis and found 51 examples of aromatic–aromatic interactions across non-hydrogen-bonded β-strands outside the iLBPs, arguing for the generality of the role played by this structural motif.     

The Role of Altered Cell–Cell Communication in Melanoma Progression

Under normal homeostasis, melanocyte growth and behaviour is tightly controlled by the surrounding keratinocytes. Keratinocytes regulate melanocyte behaviour through a complex system of paracrine growth factors and cell-cell adhesion molecules. Pathological changes, leading to development of malignant melanoma, upset this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell-cell communication molecules. In particular, there is a switch from the E-cadherin-mediated keratinocyte-melanocyte partnership to the N-cadherin-mediated melanoma-melanoma and melanoma-fibroblast interaction. Other changes include the alteration in the gap junctions formed between the melanocyte and keratinocyte. Changes in the connexin expression, in particular the loss of connexin 43, may result in a reduction or a loss of gap junctional activity, which is thought to contribute towards tumour progression. In the current review we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.     

NEUROLOGICALLY HANDICAPPED CHILDREN—The Role of the Pediatrician in Rehabilitation

In the application of the broad services now available to assist a child having a major neurologic impairment, the pediatrician occupies an important role owing to his ability to consider the problem of the handicapped child in the context of his specialized knowledge of the developmental process. He thus has a large responsibility for interpretation of the problem to the child, to the parents and to his professional colleagues and for guidance of the rehabilitation regimen within the limits of the child''s developmental readiness for new experiences.The pediatrician has the opportunity to contribute significant clinical observations which may provide stimuli for future basic research and to exercise his skill as a practitioner of preventive medicine.Goals for the future achievement of the child having a major neurologic impairment must be set realistically and with great caution.     

The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection

Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII.     

The Translesion Polymerase ζ Has Roles Dependent on and Independent of the Nuclease MUS81 and the Helicase RECQ4A in DNA Damage Repair in Arabidopsis

DNA polymerase zeta catalytic subunit REV3 is known to play an important role in the repair of DNA damage induced by cross-linking and methylating agents. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the basic polymerase activity of REV3 is essential for resistance protection against these different types of damaging agents. Interestingly, its processivity is mainly required for resistance to interstrand and intrastrand cross-linking agents, but not alkylating agents. To better define the role of REV3 in relation to other key factors involved in DNA repair, we perform epistasis analysis and show that REV3-mediated resistance to DNA-damaging agents is independent of the replication damage checkpoint kinase ataxia telangiectasia-mutated and rad3-related homolog. REV3 cooperates with the endonuclease MMS and UV-sensitive protein81 in response to interstrand cross links and alkylated bases, whereas it acts independently of the ATP-dependent DNA helicase RECQ4A. Taken together, our data show that four DNA intrastrand cross-link subpathways exist in Arabidopsis, defined by ATP-dependent DNA Helicase RECQ4A, MMS and UV-sensitive protein81, REV3, and the ATPase Radiation Sensitive Protein 5A.The DNA of all living organisms is constantly exposed to damaging factors, and therefore a number of DNA damage repair and bypass mechanisms have evolved. DNA lesions that interfere with the replication machinery constitute a particular challenge for cells (Schröpfer et al., 2014a); if not repaired in a timely manner, such damage can result in the stalling or collapse of replication forks, which in turn can lead to cell death. Furthermore, one-sided double-strand breaks (DSBs) can occur when the replication fork encounters a single-strand break. Lesions within one DNA strand, such as alkylations or DNA intrastrand cross links, can be bypassed by postreplicative repair (PRR), a process that is best understood in yeast (Saccharomyces cerevisiae). This mechanism does not lead to repair of the lesion but prevents fatal long-lasting stalling of the replication fork. PRR can be divided into two branches: the error-prone pathway and the error-free pathway (for review, see Goodman and Woodgate, 2013; Haynes et al., 2015; Jansen et al., 2015). It is known from yeast that both branches of PRR are controlled by the Radiation sensitivity protein6 (Rad6) and Mms-Ubc13 E3 ubiquitin-conjugating enzyme complexes, which ubiquitinate the replicative processivity factor Proliferating Cellular Nuclear Antigen1. The monoubiquitination of PCNA at Lys-164 by Rad6-Rad18 initiates the error-prone pathway, whereas polyubiquitination additionally requires Mms2, Ubc13, and Rad5, and triggers the error-free PRR branch (Hoege et al., 2002; Moldovan et al., 2007; Lee and Myung, 2008). There are two possible competing models postulated for the error-free bypass of lesions at the replication fork, both of which depend on template-switch mechanisms; if the lesion concerns only one of the two sister strands, the undamaged strand can be used as the template for bypassing the lesion. One of the two models features the so-called overshoot synthesis, whereby the newly synthesized strand on the undamaged parental strand is elongated further than the strand blocked by the lesion. Regression of the replication fork then leads to the formation of a special type of four-way junction called a chicken-foot structure. This regression mechanism is thought to be accomplished by helicases, such as the RecQ helicase Bloom Syndrome Protein (BLM) in humans (Croteau et al., 2014). AtRECQ4A is the respective BLM homolog in Arabidopsis (Arabidopsis thaliana), and this enzyme has the ability to regress replication forks in vitro (Hartung et al., 2007, 2008; Schröpfer et al., 2014b). The second error-free subpathway entails invasion of the newly synthesized strand on the blocked sister chromatid into the complementary newly replicated strand on the other sister chromatid. Such a step forms a displacement loop-like structure in which synthesis over the damaged region can occur. In yeast, both error-free pathways are dependent on the multifunctional protein Rad5, which is known to recruit PRR factors and also exhibits helicase activity itself (Blastyák et al., 2007). We previously identified AtRAD5A as a functional Arabidopsis homolog of Rad5 (Chen et al., 2008). Interestingly, AtRAD5A is required for efficient repair by homologous recombination via the synthesis-dependent strand-annealing mechanism, a pathway that in some steps is related to the invasion model of PRR (Mannuss et al., 2010).The error-prone pathway is based on the function of translesion synthesis (TLS) polymerases, which promote replication through DNA lesions (Prakash et al., 2005). In a mechanism termed polymerase switch, the replicative polymerase is exchanged by such a TLS polymerase at a damaged site. After incorporation of a nucleotide opposite the damaged base by the TLS polymerase, a second polymerase switch exchanges the TLS polymerase for the replicative polymerase so that replication can proceed (Prakash and Prakash, 2002; Lehmann et al., 2007). TLS polymerases possess no 5′-3′-exonuclease activity, and therefore act in a potentially mutagenic manner. Nevertheless, depending on the damage incurred and the TLS polymerase used, damage bypass can be error free (Haracska et al., 2000; McCulloch et al., 2004).Polymerases can be divided into at least six families based on their amino acid sequences and crystal structures: A, B, C, D, X, and Y. All of them share the common structure analogous to a right hand grasping DNA with palm, finger, and thumb domains (Steitz, 1999). The amino acid sequences of the finger and thumb domains of different polymerase families are highly variable, whereas the palm domains share high similarity. The palm domain forms the largest part of the polymerase active site and contains highly conserved Asp residues that have been postulated to be involved in the catalytic activity of the enzyme (Joyce and Steitz, 1995; Steitz, 1999).Most TLS polymerases belong to the Y family of polymerases, a class of specially structured enzymes that catalyze replication over damaged templates (Ohmori et al., 2001; Sale et al., 2012). Although some polymerases of the A, B, or X family can also exhibit TLS activity, this is often not their primary function (Prakash et al., 2005). DNA Polymerase Zeta (POLζ) is a B family polymerase and consists of a DNA Polymerase Zeta subunit REV3-REV7 heterodimer, in which REV3 is the catalytic subunit with its accessory subunit and processivity factor REV7 (Nelson et al., 1996). Recent studies in yeast and human cells have shown that POLζ contains two additional subunits, Pol31 and Pol32 in yeast, orthologs to human POLD2 and POLD3, which are known to be accessory subunits of the replicative polymerase POLδ (Johnson et al., 2012; Lee et al., 2014). REV3 contains three regions that are highly conserved between organisms: an N-terminal region, a REV7 binding domain, and a B family-type polymerase domain. The polymerase domain carries the six common conserved regions, I to VI (IV-II-VI-III-I-V), of which I is the most and VI the least conserved region. The A (II), B (III), and C (I) motifs, located within regions I, II, and III (Wong et al., 1988), form the active site of the enzyme, and each harbors an essential Asp residue that coordinates two catalytic metal ions. Deficiency of REV3 in mice is embryo lethal (Bemark et al., 2000; Esposito et al., 2000), and vertebrate cells depleted in REV3 show hypersensitivity to various DNA-damaging agents, including UV and ionizing irradiation, cisplatin, MMS, and mitomycin C (MMC; Sonoda et al., 2003; Sharma and Canman, 2012). In Arabidopsis, rev3 mutants exhibit no obvious phenotype under standard growth conditions, but are hypersensitive to UV-B and gamma irradiation, MMC, MMS, and cisplatin (Sakamoto et al., 2003).Our previous work demonstrated the existence of several different pathways in Arabidopsis involved in repairing the DNA damage induced by cross-linking and methylating agents. These independent pathways are defined by the ATPase RAD5A, the helicase RECQ4A, and MMS and UV-sensitive protein81 (MUS81; Mannuss et al., 2010). The structure-specific endonuclease MUS81 together with its noncatalytic subunit (Mms4 in yeast, Eme1 in Schizosaccharomyces pombe, and MMS4 or Crossover Junction Endonuclease (EME1) in humans and plants) functions in the rescue of stalled replication forks. The enzyme is able to cleave the stalled fork at the lesion site, which leads to a one-sided DSB that is repaired by homologous recombination (HR) to restore the stalled fork (Hanada et al., 2006). We previously showed that mus81 transfer DNA (T-DNA) insertion lines in Arabidopsis are highly sensitive to treatment with MMS, cisplatin, hydroxyurea, ionizing irradiation, and MMC. We also found that AtMUS81 can form a complex with its heterologous binding partners AtEME1A or AtEME1B that is able to process intricate DNA structures, such as nicked Holliday junctions, which might also form at stalled replication forks (Hartung et al., 2006; Geuting et al., 2009).In the current study, we address whether fully functional polymerase activity is required for the repair of DNA damage induced by alkylating and cross-link-inducing agents. Moreover, we sought to clarify whether REV3 cooperates with other key factors identified in Arabidopsis in the repair of these different types of damage. Indeed, it has already been shown that AtREV3 and AtRAD5A do not cooperate in the repair of such DNA damage, confirming independent pathways of error-prone and error-free PRR in plants (Wang et al., 2011). However, as plants, animals, and yeast differ in their DNA cross-link repair machinery (e.g. Mannuss et al., 2010; Knoll et al., 2012; Dangel et al., 2014; Herrmann et al., 2015), it is of particular importance to define the role of REV3 in relation to MUS81 and RECQ4A.     

The Role of Cohesiveness in the Permeability of the Spatial Assemblies of FG Nucleoporins

Nuclear pore complexes (NPCs) conduct selective, bidirectional transport across the nuclear envelope. The NPC passageway is lined by intrinsically disordered proteins that contain hydrophobic phenylalanine-glycine (FG) motifs, known as FG nucleoporins (FG nups), that play the key role in the NPC transport mechanism. Cohesive interactions among the FG nups, which arise from the combination of hydrophobic, electrostatic, and other forces, have been hypothesized to control the morphology of the assemblies of FG nups in the NPC, as well as their permeability with respect to the transport proteins. However, the role of FG nup cohesiveness is still vigorously debated. Using coarse-grained polymer theory and numerical simulations, we study the effects of cohesiveness on the selective permeability of in vitro FG nup assemblies in different geometries that have served as proxies for the morphological and transport properties of the NPC. We show that in high-density FG nup assemblies, increase in cohesiveness leads to the decrease in their permeability, in accordance with the accepted view. On the other hand, the permeability of low-density assemblies is a nonmonotonic function of the cohesiveness, and a moderate increase in cohesiveness can enhance permeability. The density- and cohesiveness-dependent effects on permeability are explained by considering the free-energy cost associated with penetrating the FG nup assemblies. We discuss the implications of these findings for the organization and function of the NPC.     

Role of the conserved Ser–Tyr–Lys triad of the SDR family in sepiapterin reductase

Sepiapterin reductase (EC 1.1.1.153; SPR) is an enzyme involved in the biosynthesis of tetrahydrobiopterin; and SPR has been identified as a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family based on its catalytic properties for exogenous carbonyl compounds and molecular structure. To examine possible differences in the catalytic sites of SPR for exogenous carbonyl compounds and the native pteridine substrates, we investigated by site-directed mutagenesis the role of the highly conserved Ser–Tyr–Lys triad (Ser and YXXXK motif) in SPR, which was shown to be the catalytic site of SDR-family enzymes. From the analysis of catalytic constants for single- and double-point mutants against the triad, Ser and YXXXK motif, in the SPR molecule, participate in the reduction of the carbonyl group of both pteridine and exogenous carbonyl compounds. The Ser and the Tyr of the triad may co-act in proton transfer and stabilization for the carbonyl group of substrates, as was demonstrated for those in the SDR family. But either the Tyr or the Ser of SPR can function alone for proton transfer to a certain extent and show low activity for both substrates.     

Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.